Industrial Biotechnology: Plant Systems, Resources and Products 9783110563306, 9783110563337, 9783110563542, 2019936646

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Mukesh Yadav, Vikas Kumar and Nirmala Sehrawat Industrial Biotechnology

Also of interest Chemical Product Technology Murzin,  ISBN ---- e-ISBN ----

Product and Process Design. Driving Innovation Harmsen, De Haan, Swinkels,  ISBN ---- e-ISBN ---- Environmental Engineering. Basic Principles Tomašić, Zelić,  ISBN ---- e-ISBN ---- Membrane Engineering. Drioli, Giorno, Macedonio (Eds.),  ISBN ---- e-ISBN ----

Industrial Biotechnology. Benvenuto,  ISBN ---- e-ISBN ----

Industrial Biotechnology Plant Systems, Resources and Products Edited by Mukesh Yadav, Vikas Kumar and Nirmala Sehrawat

Editors Dr. Mukesh Yadav Maharishi Markandeshwar (Deemed to be University) Department of Biotechnology Engineering Block-II Mullana-Ambala Haryana-133207 India [email protected] Dr. Vikas Kumar Maharishi Markandeshwar (Deemed to be University) Department of Biotechnology Engineering Block-II Mullana-Ambala Haryana-133207 India [email protected] Dr. Nirmala Sehrawat Maharishi Markandeshwar (Deemed to be University) Department of Biotechnology Engineering Block-II Mullana-Ambala Haryana-133207 India [email protected]

ISBN 978-3-11-056330-6 e-ISBN (PDF) 978-3-11-056333-7 e-ISBN (EPUB) 978-3-11-056354-2 Library of Congress Control Number: 2019936646 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.dnb.de. © 2019 Walter de Gruyter GmbH, Berlin/Boston Typesetting: Integra Software Services Pvt. Ltd. Printing and binding: CPI books GmbH, Leck Cover image: Reptile8488/iStock / Getty Images Plus www.degruyter.com

Preface Industrial biotechnology is one of the most promising approaches to fulfill the everincreasing demand of the world in terms of health, food, nutrition and other aspects of life. It offers new ways to reduce costs and create new products while protecting the environment by preventing pollution and conserving resources. The innovations in biotechnology help us to produce novel biological products. Industrial biotechnology includes use of microorganisms, plants, animal cells and enzymes to produce various products or metabolites for different industries, including food, pharmaceuticals, biodegradable plastic, biochemical industry, agricultural industry, health and diagnostics industry, and bio-fuel industry. The plant systems and resources have enormous prospective benefits to mankind. The increasing demand and continuous growth in population and undesirable climatic changes leads to decline and loss of plant resources. Therefore, there is an urgent need to develop advanced techniques for sustainable utilization of plant resources. Currently, plant biotechnology has been developed as a new age of science and technology. It is a powerful approach for the development of new plant traits and varieties. Such new varieties must be produced on a large scale to achieve commercial success as well as to satisfy the demand. Currently, the major focus of plant biotechnology is on production of secondary metabolites, valuable plant genetics improvements, germplasm conservation and production of disease-free plant verities on large scale. Plant biotechnology also works to provide the cost-effective, easily available and environment-friendly substrates in microbial biotechnology to produce microbial metabolites. This book covers the scope and relevance of plant systems and resources for the production of industrially important products or metabolites of plant origin and microbial origin as well. The plant or agricultural waste material can be used as substrate for microbial biomass and various product formations. The waste from agro-food industry can be of economic value and may be used as source of valuable metabolites. In case of plant itself, the medicinal plants are valuable sources of herbal products, and they are disappearing at a high speed. Moreover, secondary metabolites of plant origin are of biological origin, better in terms of cost, safe to consume and have more demand as compared to synthetic compounds. Plants can be transformed genetically to either introduce new traits or alter the existing traits. Higher production of metabolites, edible vaccines, chloroplast engineering, plant tissue culture and plant pigments are also important aspects of plant biotechnology in terms of industrial point of view. This book reviews the research, concise and collective information, global trends, and developments and prospects for the strategies and methodologies concerning the sustainable use of plant resources to support the expanding plan based and other dependent industries. Transgenic plants can be the cost-effective resource to produce industrially important enzymes. Further, plants provide inexpensive https://doi.org/10.1515/9783110563337-201

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Preface

production platforms for pharmaceuticals and nutraceuticals. The concept of plant-based biosensors and their practical approach have also been included in the book. We hope that this book will provide an important insight regarding the importance of plant systems and resources for the industrial side of biotechnology. More elaborative research on methodology and commercial approach is required for sustainable use of plant resources and also for conservation of plant diversity.

Contents Preface

V

Editors’ Biographies

XIII

R. S. Singh and Taranjeet Singh 1 Inulinase and pullulanase production from agro-industrial residues

1

Balwinder Singh Sooch, Yogita Lugani and Ram Sarup Singh 2 Agro-industrial lignocellulosic residues for the production of industrial enzymes 31 Amit Kumar, Diwakar Aggarwal, Mukesh Yadav, Pawan Kumar and Vikas Kumar 3 Biotechnological conversion of plant biomass into value-added products 51 Ashish Kumar Singh and Neelam Verma 4 Plants and plant-derived materials used for biosensor development

73

Pawan Kumar, Pooja Sharma, Vikas Kumar, Amit Kumar, Amit Kumar, Raj Singh and Anil K. Sharma 5 Plant resources: In vitro production, challenges and prospects of secondary Metabolites from medicinal plants 89 Navneet Kaur, Vikas Kumar, Parveen Rishi, Nirmala Sehrawat, Rahul Dilawari, Pawan Kumar and Neeraj Kumar Aggarwal 6 Phytomedicine: History, scope and future prospects 105 Diwakar Aggarwal, Anil Kumar, Amit Kumar 7 Plant tissue culture for commercial propagation of economically important plants 121 S.Vimala Devi, P.R. Kole, R. Gowthami, N. Sehrawat 8 Genetically modified plants: Developments and industrial aspects

145

Aman Verma 9 Chloroplast genetic engineering: Concept and Industrial applications Ram Kumar Pundir, Pranay Jain, Satish Kumar, Mukesh Yadav, Rajesh Kumar 10 Plant biotechnology: Industrial prospects and scopes 205 Index

219

173

List of contributing authors Chapter 1

Chapter 3

Ram Sarup Singh Carbohydrate and Protein Biotechnology Laboratory Department of Biotechnology Punjabi University Patiala 147 002 Punjab, India [email protected]

Amit Kumar Department of Biotechnology College of Natural and Computational Sciences Debre Markos University Debre Markos, Ethiopia [email protected]

Taranjeet Singh Carbohydrate and Protein Biotechnology Laboratory Department of Biotechnology Punjabi University Patiala 147 002 Punjab, India [email protected] Chapter 2 Balwinder Singh Sooch Department of Biotechnology Punjabi University Patiala-147 002 Punjab, India [email protected] Yogita Lugani Department of Biotechnology Punjabi University Patiala-147 002 Punjab, India [email protected] Ram Sarup Singh Carbohydrate and Protein Biotechnology Laboratory Department of Biotechnology Punjabi University Patiala 147 002 Punjab, India [email protected]

https://doi.org/10.1515/9783110563337-202

Diwakar Aggarwal Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana-Ambala-133207 Haryana, India [email protected] Mukesh Yadav Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala-133207 Haryana, India [email protected] Pawan Kumar Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana, India [email protected] Vikas Kumar Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana (Ambala) 133207 Haryana, India [email protected]

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List of contributing authors

Chapter 4 Ashish Kumar Singh Department of Biotechnology Punjabi University, Patiala, India And Department of Biotechnology Multani Mal Modi College, Patiala, 147001 India [email protected]

Amit Kumar Department of Biotechnology College of Natural and Computational Sciences Debre Markos University Debre Markos, Ethiopia [email protected]

Neelam Verma Department of Biotechnology Punjabi University, Patiala: 147001 India [email protected]

Anil Kumar Sharma Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala Haryana, India [email protected]

Chapter 5

Chapter 6

Pawan Kumar Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana, India [email protected]

Navneet Kaur Institute of Microbial Technology (IMTech) Chandigarh 160036, India [email protected]

Pooja Sharma Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana, India [email protected] Vikas Kumar Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana, India [email protected] Amit Kumar Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana, India [email protected]

Vikas Kumar Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana (Ambala) 133207 Haryana, India [email protected] Parveen Rishi Department of Microbiology Panjab University Chandigarh 160014, India [email protected] Nirmala Sehrawat Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana-Ambala 133207 Haryana, India [email protected] Rahul Dilawari Institute of Microbial Technology (IMTech) Chandigarh 160036, India [email protected]

List of contributing authors

Pawan Kumar Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana (Ambala) 133207 Haryana, India [email protected] Neeraj Kumar Aggarwal Department of Microbiology Kurukshetra University Kurukshetra 136119 Haryana, India [email protected] Chapter 7 Diwakar Aggarwal Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana-Ambala-133207 Haryana, India [email protected] Anil Kumar TIFAC-Center of Relevance and Excellence in Agro and Industrial Biotechnology Thapar Institute of Engineering and Technology Patiala-147001 Punjab, India [email protected] Amit Kumar Department of Biotechnology College of Natural and Computational Science Debre Markos University Debre Markos, Ethiopia [email protected] Chapter 8 Vimala Devi Sadanandam ICAR-National Bureau of Plant Genetic Resources New Delhi-110 012 India [email protected]

Pravas Ranjan Kole Protection of Plant Varieties and Farmers’ Rights Authority New Delhi-110 012 India [email protected] Rawi Gowthami ICAR-National Bureau of Plant Genetic Resources New Delhi-110 012 India [email protected] Nirmala Sehrawat Department of Biotechnology Maharishi Markandeshwar (Deemed To Be University) Mullana, Ambala Haryana 133207 India [email protected] Chapter 9 Aman Verma ICAR-Directorate of Groundnut Research Junagarh Gujarat 362001, India [email protected] Chapter 10 Ram Kumar Pundir Department of Biotechnology Ambala College of Engineering and Applied Research Devsthali, PO Sambhlakha Ambala-133101 Haryana, India [email protected] Pranay Jain Department of Biotechnology University Institute of Engineering and Technology Kurukshetra University Kurukshetra-136119 Haryana, India [email protected]

XI

XII

List of contributing authors

Satish Kumar Department of Biotechnology Ambala College of Engineering and Applied Research Devsthali, PO Sambhlakha Ambala-133101 Haryana, India [email protected] Mukesh Yadav Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala-133207 Haryana, India [email protected]

Rajesh Kumar Department of Agriculture Botany Gochar Mahavidhyalaya Rampur Maniharan Saharanpur-247451 Uttar Pradesh, India [email protected]

Editors’ Biographies Dr Mukesh Yadav is Assistant Professor at the Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana (Ambala), India. His research areas include microbial enzymes, microbial biotechnology and microbial metabolites of industrial importance. He has published research and review articles in various reputed national and international journals. He is lifetime member of several professional scientific societies and contributing to the development of society.

Dr Vikas Kumar is Assistant Professor at Maharishi Markandeshwar (Deemed to be University), Mullana (Ambala), India, and has more than four years of experience in teaching. He received his doctorate in Microbiology from Kurukshetra University, Kurukshetra, Haryana, India. He has guided several MSc, MTech and PhD students in their research work. He has published a book and more than 40 research papers and reviews in journals of national and international repute. He also serves as an associate editor and reviewer of various journals. As a researcher, his research interest includes areas like development of biological control agents, plant pathology and antimicrobial activity of natural and chemical compounds. Currently, he is working on a project which researches on role of endophytic fungi for improving the plant growth. Dr Nirmala Sehrawat is Assistant Professor at the Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana (Ambala), India. With more than nine years of teaching and research experience, she has published research and review articles in various reputed journals. Currently, she is working on a project on food grain legumes and their industrial aspects. She is an active member of several professional and scientific societies. She has been serving as an editorial board member for several national and international journals.

https://doi.org/10.1515/9783110563337-203

R. S. Singh and Taranjeet Singh

1 Inulinase and pullulanase production from agro-industrial residues Abstract: Inulinases and pullulanases have attracted the global attention of researchers owing to their vast applications in food industries. Inulinases hydrolyze the glycosidic linkages of inulin to produce fructooligosaccharides and high fructose syrup, whereas pullulanases hydrolyze the α-1,4 and α-1,6 glycosidic linkages in pullulan, starch and amylopectin to yield maltotriose, resistant starch, panose, isopanose, etc. Commercially, pullulanases are used in the starch liquefication and saccharification process, which reduces the use of glucoamylases by 50% in the industrial starch conversion process. Cost of substrate is an important factor in bioprocess economics. Therefore, many low-cost fermentation processes have been developed for the production of inulinases and pullulanases using various agro-industrial wastes. This chapter focuses on the production of inulinases and pullulanases in both the solid state and submerged fermentation (SmF) using agro-industrial residues, their purification, characterization as well as their applications. Keywords: Inulinase, pullulanases, glucoamylases, liquefication, saccharification

1.1 Introduction Inulinases, are an important class of industrial enzymes belonging to the glycoside hydrolase (GH) family 32, are characterized on the basis of their action pattern on inulin. Although inulinases have been reported from a variety of plants, animals and microorganisms, they are present in very minute quantities in plants and animals, thereby making microorganisms the best source of inulinases. Among the microorganisms, Aspergillus sp., Bacillus sp., Kluyveromyces sp., Penicillium sp., Pseudomonas sp. and Streptomyces sp. are quite efficient inulinase producers [1]. There are several advantages of microbial inulinases, such as they offer easy cultivation and genetic manipulation, high production yield, rapid multiplication, and considerable variability in biophysical and biochemical characteristics, and they are mostly inducible and extracellular in nature [2, 3]. Inulinases from fungal sources can be produced at lower substrate concentrations and have low pH and higher temperature stability, making them industrially more advantageous over other microbial sources. Inulinases have been produced in both solid state and submerged fermentations (SmFs) using a wide range of substrates and microorganisms [2, 3].

https://doi.org/10.1515/9783110563337-001

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R. S. Singh and Taranjeet Singh

On the basis of their mode of action, inulinases are classified as exoinulinases (EC 3.2.1.80) and endoinulinases (3.2.1.7). Exoinulinases perform sequential degradation from the non-reducing end of the inulin, releasing fructose with a molecule of glucose, whereas endoinulinases act arbitrarily on the internal β-2,1glycosidic linkages of inulin to produce fructooligosaccharides of varied chain lengths. The exoacting and endoacting natures of inulinases mainly depend on the source of the enzyme. Inulinases from fungal sources are generally exoacting. However, in some cases, fungal strains like Aspergillus sp., Chrysosporium pannorum and Penicillium rugulosum, and the yeast strain Cryptococcus aureus produce both exo- and endoinulinases [1, 2]. Although most of the microorganisms produce extracellular inulinases, in some strains the enzyme location is intracellular [4, 5], and yet some other microbial species produce both extra- and intracellular inulinases [6]. Inulinases are used for the production of high fructose syrup, fructooligosaccharides, bioethanol, single-cell oil, single-cell proteins and other beneficial bio-products, and hence they play a predominant role in various food and pharmaceutical industries [7–10]. Pullulanase-debranching enzymes belong to the GH 13 family, which hydrolyzes the glycosidic linkages in pullulan and other branched polysaccharides. Pullulanases degrade pullulan, β-limit dextrin and amylopectin, and the detailed mechanisms of substrate degradation and the final end products are different in each case. Based on substrate specificity and catalytic reaction end products, pullulan-hydrolyzing enzymes have been classified into three types: pullulanases (types I and II), pullulan hydrolases (types I, II and III) and glucoamylases. Pullulanases (types I and II) hydrolyze α-1,6 linkages in pullulan and starch to produce maltotriose. Pullulan hydrolases type I (neopullulanase) and type II (isopullulanase) break the α-1,4 linkages of pullulan to release panose and isopanose, whereas pullulan hydrolase type III breaks both α-1,4 and α-1,6 linkages in pullulan to release maltotriose, maltose and pentose. However, glucoamylase hydrolyzes α-1,4 linkages from the non-reducing end of pullulan to yield glucose as a product.

1.2 Microbial sources of inulinases Microorganisms from various microbial groups like filamentous fungi, bacteria and yeast strains have been successfully used for inulinase production (Figure 1.1). Generally, filamentous fungi are preferred over bacterial and yeast strains because of their ability to grow at low pH and high temperatures, which reduce the chances of contamination and secondary product formation. Moreover, inulinase-producing fungi can also be cultivated at low-value substrates, thus making the fermentation process more cost-effective. Among the filamentous fungi, Aspergillus sp., Penicillium sp. and Cladosporium sp. have been reported to be prolific inulinase producers.

1 Inulinase and pullulanase production from agro-industrial residues

3

Figure 1.1: Inulinase-producing microorganisms.

Aspergillius niger, A. niveus, A. ficuum, A. tamari, A. terreus and A. tubingensis are potent inulinase-producing aspergilli, whereas Penicillium subrubescens, P. expansum, P. purpurogenum, P. rugulosum and P. trzebinskii have been reported as potent inulinase-producing penicilli. Moreover, among the various species of Cladosporium, C. cladosporioides and C. phoenici are very effective inulinase producers [2].

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R. S. Singh and Taranjeet Singh

Bacterial strains like Bacillus sp., Streptomyces sp., Xanthomonas sp. and Clostridium sp. are used for inulinase production because of their ability to survive under unfavorable conditions like high temperature, alkalinity, acidity and salinity. Therefore, isolation of bacterial strains producing high quantities of thermostable inulinases is in great demand for industrial applications. Recently, bacterial strains like Bacillus sp. [11], B. safensis [12], Acinetobacter baumannii [13] and B. centrosporus [14] have been reported as new inulinase producers. Some extremophilic bacteria like Streptomyces sp., Bacillus sp., Clostridium thermoautotrophicum, Xanthomonas oryzae and Sphingobacterium sp. have also been reported to produce characteristically distinguishable inulinases [3]. Owing to their ease of handling and fast growth rates, yeast strains are being preferred over fungal strains for inulinase production. Among the different yeast strains, Kluyveromyces sp., Pichia sp. and Candida sp. are potent inulinase producers. Kluyveromyces marxianus is a well-known inulinase producer, whereas some of the marine-derived yeasts, such as Meyerozyma guilliermondii [15], Cryptococcus aureus [16] and Zygosaccharomyces cerevisiae [17], are efficient inulinase producers. Recently, a few yeast strains like Zygosaccharomyces, Gordonia, Hanseniaspora, Torulosopra, Saccharomyces, Metschnikowia and Lachanceahave have also been screened for inulinase production [18].

1.3 Production of inulinases from agro-industrial residues A wide range of microorganisms from filamentous fungi, bacteria, yeast and actinomycetes have been reported for inulinase production in solid-state fermentation (SSF) using agro-industrial residues as the substrate [2, 3]. The substrate and the type of microorganisms used for the production of inulinases play a vital role in the localization of the enzyme, its mode of action as well as its yield.

1.3.1 Submerged fermentation (SmF) SmF is one of the most efficacious and finest techniques used for inulinase production at the industrial scale. It has been practiced since the past few centuries due to its numerous advantages like ease of process control, sterilization process, media homogeneity and appropriate heat transfer system. [19]. Although various substrates have been used for the production of inulin hydrolytic enzymes, inulin and inulin-rich plant extracts are mainly used in SmF. Raw inulin

1 Inulinase and pullulanase production from agro-industrial residues

5

extracted from tubers of Asparagus offcinalis [20], Asparagus racemosus [21] and dahlia [22] have been used as a substrate for inulinase production in shake-flask fermentations. The degree of polymerization (DP) of inulin also plays a pivotal role in inulinase production. In a comparative study, inulinase production by A. niger was observed using raw and dried garlic inulin as a substrate [23]. It has been reported that dried garlic produces lower enzyme yield than raw garlic. This is because fresh raw garlic has a higher inulin content than dried garlic, where inulin may get degraded because of storage and seasonal changes. There are various key factors that can result in successful SmF like carbon and nitrogen sources, pH, temperature, aeration and agitation. Inulin can be either used in the pure form or mixed with other natural substrates for the production of inulinases (Table 1.1). It acts as an inducer as well as a sole carbon source for different classes of microbes. Other substrates like glucose, sucrose, lactose, maltose and fructan can also be used for inulinase production [1]. Angel et al. [24] formulated a medium to study the effects of different carbon sources – inulin, sucrose, xylose, fructose and glucose on inulinase production from Bacillus sp., Pseudomonas sp., Lactobacillus sp. and Achromobacter sp. Although inulin is considered the most suitable carbon source for inulinase production from all the bacterial strains under SmF, modified substrates such as caproyl and cholesteryl derivatives of dahlia inulin and octadecanoylsucrose derivatives have also been used as a carbon source for inulinase production [25, 26]. Nitrogen source is the second important medium constituent used for enhancing inulinase production. Various complex nitrogen sources (peptone, beef extract, yeast extract, meat extract, corn steep liquor, urea, etc.) and inorganic nitrogen sources (NH4Cl, (NH4)2SO4, (NH4)2H2PO4, (NH4)2HPO4, NaNO3, KNO3, etc.) have been extensively used for inulinase production [10]. Complex nitrogen sources result in better inulinase production than inorganic nitrogensources [21, 27, 28]. Despite acid being usually liberated in the medium after the utilization of ammonium salts and high acidic conditions inhibit the growth and synthesis of inulinases. Ammonium salts were found necessary to induce inulinase biosynthesis from B. polymyxa and B. subtilis [29]. Organic (casein hydrolysate) and inorganic (like (NH4)2SO4) nitrogen sources showed synergism in stimulating bacterial growth and inulinase biosynthesis [30]. Metal ions like Mn2+, Ca2+, Zn2+, Mg2+, Fe2+ and K+ had a positive influence on microbial inulinase production [1, 2]. Fungal inulinase producers are generally cultivated in a fermentation medium at the pH range of 4.5–7.0, bacterial strains at the pH range of 4.8–7.0 and yeast strains at the pH range of 4.4–6.5. The optimal temperature for bacterial and fungal strains lies in the mesophilic to thermophilic range (28–60 °C), and for yeasts strains, it lies in the mesophilic range (30–45 °C) [25]. Most of the reports describe the mesophilic range to be more suitable for inulinase production, but there are a few thermophilic inulinase producers too, including Bacillus smithii, Pseudomonas sp., Rhizoctonia solani and Streptomyces sp. [2, 3].

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Table 1.1: Microbial production of inulinases in solid-state fermentation (SSF) and submerged fermentation (SmF). Microorganism

Substrate used

Type of fermentation

Type of inulinase

Yield (IU/mL)

Aspergillius tritici

Asparagus officinalis extract

SmF

Endoinulinase

.

[]

Aspergillus niger

Sucrose

SmF

Exo- & endoinulinase



[]

Banana peel

SSF

Exoinulinase



[]

Dandelion taproot extract

SmF

Endoinulinase



[]

Helianthus annuus SSF root tubers & Lactuca sativa roots

Exoinulinase



[]

Pure inulin

SmF

Exo- & endoinulinase

 IU/ mga

Pure inulin & wheat bran

SmF

Exoinulinase

.

[]

Pure inulin

SmF

Exoinulinase

.

[]

Agrowaste extract

SmF

Exoinulinase

.

[]

Aspergillus terreus

Artichoke leaves

SSF

Exoinulinase

.

[]

Bacillus safensis

Pure inulin

SmF

Endoinulinase

.

[]

Asparagus racemosus root tubers extract

SmF

Endoinulinase

.

[]

Bacillus smithii

Pure inulin

SmF

Endoinulinase

.

[]

Brevibacillus centrosporus

Jerusalem artichoke tubers extract

SmF

Exoinulinase

.

[]

Chrysosporium pannorum

Pure inulin

SmF

Exo- & endoinulinase



[]

Cryptococcus aureus

Pure inulin

SmF

Exo&endoinulinase



[]

Wheat bran & rice husk

SSF

Exo- & endoinulinase

.

[]

Aspergillus tamarii

Reference

[]

1 Inulinase and pullulanase production from agro-industrial residues

7

Table 1.1 (continued ) Microorganism

Substrate used

Type of fermentation

Type of inulinase

Yield (IU/mL)

K. marxianus

Sugarcane molasses

SmF

Exoinulinase



[]

Dahlia tubers extract

SmF

Exoinulinase

.

[]

Dahlia tubers extract

SmF

Exoinulinase

.

[]

Asparagus racemosus root tubers extract

SmF

Exoinulinase

.

[]

Asparagus officinalis root tubers extract

SmF

Exoinulinase

.

[]

K. marxianus var. marxianus

Pressmud

SSF

Exoinulinase

.

[]

Penicillium citrinum

Pure inulin

SmF

Exoinulinase



[]

Pure inulin

SmF

Exoinulinase

.

[]

Penicillium oxalicum

Lactose

SmF

Exoinulinase

.

[, ]

Penicillium rugulosum

Copra waste

SSF

Exoinulinase



[]

Pure inulin

SmF

Exo- & endoinulinase

. IU/mga

[]

Penicillium sp.

Dahlia tubers extract

SmF

Endoinulinase

.

[]

Pseudomonas sp.

Chicory roots powder SmF

Endoinulinase



[]

Rhizopus sp.

Pure inulin

SmF

Endoinulinase

. IU/ mga

[]

Rhodotorula glutinis

Leek powder

SmF

Exoinulinase

.

[]

Saccharomyces sp.

Wheat bran

SSF

Exoinulinase

.

[]

Streptomyces sp.

Jerusalem artichoke powder

SmF

Endoinulinase

.

[]

Copra waste

SSF

Exoinulinase



[]

Jerusalem artichoke powder

SmF

Endoinulinase



[]

Trichoderma viride

Reference

8

R. S. Singh and Taranjeet Singh

Table 1.1 (continued ) Microorganism

Substrate used

Type of fermentation

Type of inulinase

Yield (IU/mL)

Ulocladium atrum

Pure inulin

SmF

Exoinulinase



[]

Xanthomonas sp.

Chicory roots powder SmF

Endoinulinase



[]

Dahlia tubers extract

SmF

Endoinulinase



[]

Pure inulin

SmF

Endoinulinase

.

[]

Yarrowia lipolytica a

Reference

Specific activity of endoinulinase in crude extract

1.3.2 Solid-state fermentation (SSF) Recently, SSF has become the most prominent and efficient method for inulinase production at the industrial scale. SSF has become an attractive alternative to SmF due to its numerous advantages, such as use of low-value substrates, which makes the bioprocess cost-effective; enhances product concentration; better oxygen circulation; providing physical support to growing cells; higher yield and recovery; environmental friendly nature; and reduced energy requirement and water consumption [64]. Various factors like type of substrate, moisture, water activity, pH, temperature, aeration and agitation are important effective SSF. Many agro-industrial residues and inulin-rich plant parts, such as wheat bran, rice bran, pressmud, sugarcane bagasse, chicory leaves and roots, artichoke leaves, garlic and onion peels, have been used as substrates for inulinase production in SSF using different microbial strains (Table 1.1). Both high and low moisture levels are vulnerable for inulinase production during SSF. Because yeast and fungal strains can grow at a low moisture level, they can be seamlessly used in SSF. Bacterial strains like Staphylococcus sp. [65], Xanthomonas campestris pv. phaseoli [66], Streptomyces sp. [67] and P. rugulosum [51] and fungal strains like Kluyveromyces sp. [19], Aspergillus niger [68], A. parasiticus [69] and Penicillium oxalicum [70] have also been used for inulinase production under SSF. Inulinase yield of 78.29 ± 0.13 U/gds was obtained from Saccharomyces sp. when wheat bran was used as a substrate in SSF [70]. Mostly inulinase production from fungal strains is reported in the acidic range (4.5–5.5) with the exception of A. terreus, which produces inulinase at pH 8.5 [71]. However, bacterial strains are normally resistant to acidic conditions and grow well in the neutral range of pH 6.5–7.5.

1 Inulinase and pullulanase production from agro-industrial residues

9

1.4 Purification and characteristics of inulinases To determine the physicochemical characteristics of an enzyme, its purification and characterization are mandatory. Currently, various purification techniques like salt or solvent precipitation, ion-exchange chromatography, affinity chromatography, size-exclusion chromatography, hydrophobic-interaction chromatography and ultrafiltration are used for inulinase purification. Most of the inulinases are extracellular in nature; for the purification of intracellular inulinases, an additional step of cell-wall disruption is required, followed by the techniques mentioned above. The purification techniques and characteristics of some inulinases are listed in Table 1.2. Different inulinases have different hydrolytic actions (exoor endoacting) and structural conformations depending on their source and growth conditions. Five exoinulinases (Exo-I, II, III, IV and V) and two endoinulinases (Endo-I and II) from A. ficuum were purified using ammonium sulfate precipitation, ion-exchange chromatography and fast protein liquid chromatography [6]. Similarly, three exoinulinases and two endoinulinases from A. ficuum JNSP506 were purified using ion-exchange chromatography and gel-filtration chromatography [72]. Recently, three active forms of inulinases (INI, INII and INIII) from Ulocladium atrum were purified by ammonium sulfate and ion-exchange chromatography. Inulinase (INI) was resolved into INIa, INIb and INIc by chromatographic separation with 3.43-fold purification [61]. In the case of bacterial inulinases, salt precipitation shows better results than solvent precipitation as some of the organic solvents have been reported to repress the enzyme. For salt precipitation, generally ammonium sulfate is used. In the case of B. smithii, ammonium sulfate and ion-exchange chromatography were used for the purification of thermostable endoinulinases [73]. Most of the bacterial inulinases are purified by ion-exchange and size-exclusion chromatography, but there are a few reports on inulinase purification by affinity chromatography. For example, recombinant inulinase from P. polymyxa and Sphingomonas sp. have been purified using Ni2 + -NTA affinity chromatography [74, 75]. Column chromatography using DEAE Sepharose and Sephacryl-200 was used for the purification of inulinase (Inu 2 and Inu 3) from Rhiizopus oligosporus [76]. In another case, ion-exchange and gel-exclusion chromatography were used for the purification of one endoinulinase and two exoinulinases (F2 and F3) from Chrysosporium pannorum [77, 78]. The conformational alternations of a biocatalyst can be understood by its molecular weight (Mr) and the Michaelis–Menten constant (Km and Vmax). The molecular weight of fungal inulinases mainly ranges between 30 and 175 KDa and that of bacterial inulinases between 45 and 600 KDa [2, 3]. Km and Vmax of purified bacterial and fungal inulinases show considerable variations. Metal ions like Fe3+, Mn2+, Cu2+, Mg2+, Co2+, Na+ and Ag+ may either stimulate or inhibit enzyme catalysis by affecting their premolecular organization [2, 3].

pH Temperature pH (°C)  .–.

 NS  NS

 NS

 .–.

.

. .

.

.

Specific activity (IU/mg)



InuB: . InuLC: .

NS

NS

Purification techniques

(NH)SO precipitation, HiTrap-QFF & Capto-MMC column chromatography

IEC on DEAE cellulose

Affinity chromatography on Ni+-NTA

Affinity chromatography on Ni+-NTA sepharose

Paenibacillus sp. D

Lactobacillus paracasei

Aspergillus niger

Kluyveromyces cicerisporus

Kinetics

Characteristics



NS

NS NS



.a

.a

.a .a

.a

c

.c

.c .c

.b

Temperature Km Vmax (°C) (mg/mL) (mg/min)

Stability

Microorganism

Optimum

Table 1.2: Purification and characterization of inulinases.

NS

NS

NS NS

NS

. .

Cu, . Co+, Fe+

NS

NS NS

Hg+, Fe+, . Cu+

Ca+

Mr (KDa)

Inhibitor

Activator

Metal ions

[]

[]

[]

[]

Reference

10 R. S. Singh and Taranjeet Singh

 .–.

 NS

 .–.

 .–.  .–.

.

.

.

. .

.d

NS

.

Inu :  Inu : 

(NH)SO precipitation, dialysis, Ni+-NTA affinity chromatography, Sephacryl S-, DEAE sepharose column

Affinity chromatography on Ni+-NTA sepharose

Affinity chromatography on Ni+-NTA

IEC on DEAE Sepharose & Sephacryl S- CL-B

Aspergillus niger

Aspergillus niger

Sphingomonas sp.

Rhizopus oligosporus

 .–.

.

.d

(NH)SO precipitation, dialysis, Ni+-NTA affinity chromatography, Sephacryl S-, DEAE sepharose column

Kluyveromyces marxianus

 

–

NS





.f .

.

.a

.

.

NS NS

.c

.c

.e

.e

NS NS

NS

NS

NS

NS



.

.

Ni+, Cu+, Fe+, Co+, Hg+

 

Hg+,  Ag+, Mn+,

NS

NS

NS

(continued )

[]

[]

[]

[]

[]

1 Inulinase and pullulanase production from agro-industrial residues

11

Streptomyces sp.

(NH)SO precipitation, IEC on Macro-prep DEAE, GEC on Sephacryl S-, HIC on t-Butyl, Hydroxyapatite Bio-Gel HTP

.

Affinity . chromatography on Ni+-NTA, GEC on Sephacryl S-

Paenibacillus polymyxa

.

.

NS

.

(NH) SO precipitation, dialysis, ion-exchange chromatography

Ulocladium atrum

 .–.

Kinetics

Characteristics

–

–

NS

NS

.a

.a

NS

NS

c

.c

NS

NS

Temperature Km Vmax (°C) (mg/mL) (mg/min)

Stability

 .–.

NS NS

 NS

.

.g

Penicillium sp.

Ethanol precipitation, ion-exchange chromatography

Purification techniques

Microorganism

pH Temperature pH (°C)

Optimum

Specific activity (IU/mg)

Table 1.2 (continued )

Hg+

Mg+, Mn+, Co+



Co+,  Cu+, Ni+

NS



Mr (KDa)

Zn+, Fe+, Mg+

Fe+, Mn+, Ba +, Co+, Mg+

Hg+

Mn+, Ca+

NS

Inhibitor

Activator

Metal ions

[]

[]

[]

[]

Reference

12 R. S. Singh and Taranjeet Singh

niger

terreus

A.

A.

.

.

.

Ultrafiltration, IEC . on CM Sepharose & Phenyl Sepharose, GEC on Sephadex G-

Three-phase partitioning

(NH) SO & solvent precipitation, dialysis, GEC on Sephadex G-, IEC on DEAE cellulose

.

.

.

Ultrafiltration, IEC  on DEAE Sepharose & QSepharose, GEC on Sephadex  PG

 ISO brightness units as compared to wild-type wood); no observed phenotypic differences

[]

152

S.Vimala Devi et al.

Table 8.2 (continued ) Crop

Trait

Gene

Result

Reference

Pinus radiata Lignin content and composition

HCT RNAi

Decreased (%) lignin content; predominant deposition of -hydroxyphenyl units (% of wild-type); increased resinols; reduced dibenzodioxocins; presence of glycerol end groups

[]

Aspen

Lignin content

CL antisense

Decreased (%) lignin content; increased cellulose content

[]

Chinese white poplar

Lignin content

CL antisense

Decreased (%) lignin content; unchanged holocellulose content; red brown coloration

[]

Populus tremula × Populus alba

Lignin content

CL antisense

Decreased (%) lignin content; reddish-brown discolored wood

[]

Aspen

Lignin content and composition

CL × CALDH antisense CL and sense CALDH; multigene cotransformation

Decreased (%) lignin content; increased S:G (%); increased cellulose (%); accelerated cell maturation in stem secondary xylem

[]

Poplar

Lignin content and composition

CAD antisense

Unchanged lignin content; increased aldehyde

[]

Flax

Lignin content and composition

CAD RNAi

Decreased lignin content; changed lignin composition; reduction in the pectin and hemicellulose contents; improved mechanical properties; increased Young’s modulus (% higher); reduced (twofold) resistance of the transgenic lines to Fusarium oxysporum

[]

Maize

Lignin content and composition

CAD RNAi

Unchanged lignin content, slightly decreased S:G ratio and increased cellulose and arabinoxylan contents in stems; reduced lignin content and cell wall polysaccharides in midrib; midribs and stems more degradable than wild-type plants

[]

8 Genetically modified plants: Developments and industrial aspects

153

8.3.3 Timber Timber industry entirely depends on the forest tree species as the raw material source. Timber density – an indicator of wood quality – is amenable to improvement using gene manipulation [38]. Increase in the stiffness of core wood (Pinus radiate), reduced grain spiraling and desirable trunk shape are the traits that can be modified to improve the value of timber [47]. This work has been initiated with the genetic mechanism that controls timber quality like cellulose synthesis, microfibril angle, fiber cell length, cell wall thickness and early wood–latewood transitions. In addition, transgenic for resistance to pest, diseases and herbicide tolerance toward abiotic stress(freeze tolerance in eucalyptus) are important traits on which work is in progress in several research institutes for value addition to timber species[52,53].

8.3.4 Biofuels As an alternative to the depleting fossil fuels, the first-generation biofuels that were made using agricultural feedstocks play a significant role in establishing the basic infrastructure for biofuel production processes and in producing a part of transport fuels. However, in several countries the competition with food crops for land use and the food security mission has eventually led to the second-generation (based on the non-food crops) and the third-generation (based on microalgae) biofuels [54]. Sequential and spatial expression of fatty acid and lipid biosynthetic genes are related with the storage lipids accumulation in the seeds of oil plants [55]. Genetic alterations of plants by transgenic technology help in developing sustainable bio-feedstocks for biofuels production. Active oilseed feedstocks can be genetically modified to yield higher oil content and optimal fatty acid composition. This can be an effective strategy to improve the yield and fuel properties of biodiesel [56]. Furthermore, the most viable option will be the lignocellulosic biomass from crops (corn stover, wheat straw, switch grass, Miscanthus, canary grass, giant reed, alfalfa, sweet sorghum and Napier grass) and various fast-growing trees (poplars, willows and Eucalyptus) for the production of ethanol[57]. In the oil-bearing plant species, the genes involved in the oil biosynthesis pathway is well studied and found that diacylglycerol acyltransferase (DGAT), FAD gene and others play a key role in oil production [58]. Several studies have shown that overexpression of the rate-limiting enzyme in developing seeds can enhance their oil content up to 25% (w/w). Also, the antisense technology obstructs the ability of plants to break down the fatty acids and caused an increase in oil content of plant leaves under assured growth conditions [59]. Among the non-oil-bearing source, the accessibility to renewable cellulosic feedstock is almost indefinite around the globe. Moreover, the processing of lignocellulose

154

S.Vimala Devi et al.

into biofuel and fermentable sugars needs costly and harsh pretreatments, which further reduce the cost-effectiveness [60]. The structural genes that control the cellulose or lignin production and various transcription factors acting as regulators of lignin or cellulose synthesis can be the option for genetic modification. Earlier reports highlighted that genetic alteration of the enzymes involved in lignin biosynthesis pathway specifically reduces the necessity of pretreatment processes for fermentable sugars production. For example, downregulation of six different enzymes regulating lignin biosynthetic pathway in alfalfa – C3H (4-hydroxycinnamate3-hydroxylase), C4H (cinnamate 4-hydroxylase), CCoA-OMT (S-adenosyl-methioninecaffeoyl-CoA/5hydroxyferuloyl-CoA-O-methyltransferase), HCT (hydroxyl cinnamoyltransferase), COMT (caffeate-O-methyltransferase) or F5H (ferulate 5-hydroxylase) – can either reduce or abolish the needs of chemical pretreatment for the production of fermentable sugars [54,61]. The other options can be the gene regulation of the substrate-disrupting factors or modifying the features of cellulose itself for costeffective processing. Table 8.3. provides few examples where the modification in non-oilseed bearing crops has resulted in reduced lignin or modified cellulose, which ease the bioethanol production process industrially.

8.3.5 Pharmaceutical products Recent biotechnological techniques have attracted greater attention of researchers toward producing new more effective therapeutic agents by using different botanical resources. Among the different biotechnological techniques, genetic engineering of plants is one of the most desired methods than can facilitate the production of pharmacologically active proteins like blood product substitutes, vaccines, mammalian antibodies, hormones and various therapeutic agents [74, 75]. The production of proficient biopharmaceutical compounds in plants involves appropriate selection of the host plant and suitable gene expression system such as either a food crop or a non-food crop. Commercialization of plant-based biopharmaceuticals should strictly adhere to product safety issues related to the pharmaceutical workers, general public and patients [76]. This should be given high priority along with the proper control of regulatory errors during pharmaceutical compound production from plants. Plants have immense potential to produce useful compounds for mankind. Therefore, plant resources can act as a vital production system for various important biopharmaceutical compounds [77]. Transgenic plants are most commonly used to produce pharmaceuticals usually known as plant-manufactured pharmaceuticals (PMPs).Plants are an inexpensive source of protein. Moreover, plants can be easily transformed and have significant potential to produce biopharmaceutical proteins and peptides [78]. Earlier reports recognized the plants for its huge diversity and effectiveness of chemicals (cocaine, salicylic acid, taxol, morphine, etc.) having prevailing effects on human health and

8 Genetically modified plants: Developments and industrial aspects

155

Table 8.3: Milestones for the development of transgenic plants for biofuel industry. Crops

Traits

Gene

Result

References

Populus

Modification of the lignin biosynthetic machinery

-Coumarate CoA ligase (CL), cinnamyl alcohol dehydrogenase (CAD), caffeic acid -omethyltransferase (COMT), s-adenosylmethionine caffeoylCoA (CCoAOMT), ferulate -hydroxylase (FH)

Different levels of lignin reduction

[]

Lignin modification

Overexpression of the pine cytosolic gene glutamine synthetase (GSa)

Reduced lignin content

[]

Lignin modification

Ectopic expression of the Eucalyptus egmyb gene

Reduced lignin content

[]

Lignin modification

Downregulation of the CL, COMT and CAD genes

Reduced lignin content

[]

Lignin modification

Overexpression of the RR-MYB transcription factor

Reduced lignin content

[]

Switch grass

Lignin modification

Overexpresses the maize Corngrass (Cg) microRNA

Locks the plant in the juvenile stage has reduced lignin levels, % more starch and higher saccharification efficiency

[]

Sugarcane

Lignin modification

Downregulation of the sugarcane caffeic acid O-methyltransferase (COMT) gene

Reduction of the recalcitrance of biomass by reducing lignin content

[]

Switch grass

Lignin modification

RNA interference of PvCL

Reduced lignin content by % with decreased guaiacyl unit composition and improved fermentable sugar yields

[]

156

S.Vimala Devi et al.

Table 8.3 (continued ) Crops

Traits

Gene

Result

References

Populus tremuloides

Cellulose modification

Over expression of an aspen secondary wallassociated cellulose synthase (ptdcesa)

Easy breakdown of cellulose in fermentation process

[]

Sweet sorghum

Altered lignin content

Manipulating the expression of caffeoylcoa-Omethyltransferase (CCOAOMT)

Lignin modification for use in ethanol production

[]

Alfalfa

Lignin modification

Downregulation of -coumarate -hydroxylase (CH)

Dramatic shift in the lignin profile and consequent altered lignin structure, causing improved digestibility

[]

Switch grass

Lignin modification

Overexpression of mir involved in the suppression of squamosa promoter binding protein like (SPL) genes

Increase in overall biomass accumulation coupled with the potential to increase saccharification efficiency

[]

Several species

Lignin modification

Over expression of RR- MYB transcription factors

Repress lignin biosynthesis in several species

[]

Switch grass

Improvement of lignocellulosic feedstocks

Overexpression of pvmyb

Threefold increase in hydrolysis efficiency (.-fold)

[]

Maize

Plant oil

Fungal lactase gene (EMBL accession No. U)

Increased expression of oil

[]

Switchgrass

Lignin biosynthetic pathway

Downregulation of the caffeic acid -Omethyltransferase EC ... (COMT) gene

Cell walls of transgenic plants released more constituent sugars

[]

Maize

In-planta expression of endocellulases

Glycoside hydrolase family  endocellulase, E (CelA), from Acidothermus cellulolyticus

Will allow the enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion

[]

8 Genetically modified plants: Developments and industrial aspects

157

Table 8.3 (continued ) Crops

Traits

Gene

Result

References

Maize

Enzyme recovery

E cellulase, an endobeta-,-glucanase from A. cellulolyticus

Recovery of commercial amounts of enzyme at levels greater than % total soluble protein (TSP) in single seed

[]

Sugarcane

Self-processing

Microbial genes that produce cellulosedegrading enzymes to produce

To produce selfprocessing plants

[]

physiology [79]. In recent years, plants have been significantly used in the production of animal and human oral vaccines, particularly in the developing countries where traditional vaccines are mostly used [78, 80].

8.3.6 Antibodies Plant systems have been successfully explored for the expression and development of different antibodies and their derivatives. Antibodies are large and multifaceted proteins. They have enormous ability to recognize and bind to their specific molecular targets. Normally, the plants do not produce antibodies, but several studies reported that the transgenic plants expressing antibody encoding genes can form functional antibodies [81, 82]. Monoclonal antibodies are one of the significant developments of biotechnology. These are also crucial products for both diagnostic purposes and therapeutic uses. Mice have also been used to produce traditional therapeutic monoclonal antibodies. But these proteins were voluntarily recognized as foreign by the human immune system. This rigorously restricts the use of these monoclonal antibodies for therapeutic use, especially with recurring dosing. The incidence of neutralizing antibodies that inactivate the drug often precluded further therapeutic use even in the absence of serum sickness or anaphylaxis [83]. But the recombinant technologies have allowed the replacement of murine antibodies with partially humanized or chimeric antibodies. This helped in the production of fully human antibodies. Human antibodies can be obtained from mice carrying human immunoglobulin genes. These can also be produced using yeast or other gene-expression array technologies. Moreover, the recombinant technology may be used to develop an antibody gene with higher binding affinity (affinity maturation). It has also been demonstrated that the currently available recombinant antibodies exhibit increased biological activity

158

S.Vimala Devi et al.

and reduced immunogenic response as compared with the earlier monoclonal antibodies [84]. Various genetic transformation studies successfully explored the immense potential of plants to produce various recombinant proteins, such as pharmaceuticals and industrial proteins, and other secondary metabolites under different phases of clinical trials presented in Table 8.4. At present, several crops are being genetically altered to produce hormones, enzymes, vaccines, monoclonal antibodies and anticoagulant factors for the diagnosis, treatment or prevention of diseases in animals and human [75, 78, 85].

Table 8.4: Utilization of transgenic plants for the production of various kinds of antibodies. Plant

Genetic transformation system

Protein

Result

Reference

Agrobacteriummediated transformation

IgG specific for human creatine kinase

IgG assembly and secretion

[]

Agrobacteriummediated transformation

IgG (Guy’s ) specific for S.mutans surface protein (SA I/II)

Comparison of glycosylation in plantand animal-derived IgG

[]

Agrobacteriummediated transformation

IgG (Guy’s ) specific for S.mutans surface protein (SA I/II)

Synthesis of fulllength IgG

Agrobacteriummediated transformation

Hybrid sIgA-G specific Synthesis of secretory for S.mutans antigen II immunoglobin for treatment of dental caries

Immunoglobulin Tobacco

[, ]

[, ]

Single-chain Fv fragments Potato

Agrobacteriummediated transformation

Phytochrome binding scFv

Accumulation and storage of protein in tubers

[]

Tobacco

Agrobacteriummediated transformation

scFv of IgG from mouse B-cell lymphoma

Treatment of nonHodgkin’s lymphoma

[]

Cereals

Particle bombardment

scFvT. against carcinoembryogenic antigen

Production of tumorassociated marker antigen

[]

8 Genetically modified plants: Developments and industrial aspects

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8.3.7 Vaccines The vaccines produced in plants can express the entire selected proteins. Therefore, the use of precise DNA encoding only specific antigenic sequences from bacteria, parasites and pathogenic viruses has received significant consideration [92]. Many food plants such as asparagus, apple, alfalfa, banana, cabbage, barley, canola, carrots, cantaloupe, cranberry, cauliflower, cucumber, flax, eggplant, grape, lettuce, kiwi, lupins, melon, maize, pea, papaya, peanut, plum, pepper, potato, rice, raspberry, soybean, service berry, squash, sweet potato, sugar beet, strawberry, sunflower, sugarcane, tomato, wheat and walnut have been transformed to produce valuable vaccines (Table 8.5) and biopharmaceuticals (Table 8.6) [93].

8.3.8 Other industrial oils/fatty acids Oil-bearing tree species produce oils or hydroxyl fatty acids. These can be used to produce super quality lubricants for jet engines and also in the manufacturing of cosmetics, shampoo, antifungal compounds, plastics, paints and coating. Castor is one of such examples that are highly required in the industrial sector. However, it contains a highly poisonous compound ricin, which is more toxic than the snake venom. Castor can be genetically engineered to produce seed oils with epoxy fatty acids as vernolic acid (12, 13-epoxy-cis-9-octadecenoic), which is produced in Vernonia galamensis [110]. Epoxy oils form tremendous coatings on plastics and steel. It also helps in the preparation of improved lubricants, paints and coatings. This kind of genetic alterations can yield plant that does not produce ricin, and thus can revitalize the interest in this crop. Genetic modifications of crop plants facilitate to “knock out” the genes responsible for production of ricin, ricinine and CB-1A [111]. Castor meals free of ricin are significant in producing rations and animal feeds. Another important oil-bearing crop is Jatropha. RNA silencing of curcin gene can inhibit production of toxin and further add economic fodder value to biofuel feedstock. Enhanced expression of the stearoylacyl carrier protein desaturase and JcERF genes in Jatropha can exhibit increased seed oil and improved resistance toward salt stress and frost. Guar or cluster bean (Cyamopsis tetragonoloba), an annual legume, is an excellent source of guar gum. This has made guar a precious commodity for export. Genetic modification of ADH (alcohol dehydrogenase) gene in this valuable crop may lead to enhanced ethanol production [112]. Calendic acid is oxidatively more unstable as compared to linolenic acid. Hence, it helps in improving the drying properties of coating applications [113]. The concentration of calendic acid is lower in soybean (20–25%) than in marigold (55%). Many other biochemical pathways are actively engineered to produce useful molecules for the chemical industry. But most of them are still at R&D stage such as

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Table 8.5: Production of antigens to prepare vaccines for specific disease in transgenic plants through genetic transformation systems. Plant species Genetic transformation system

Disease

Vaccine antigens

Black-eyed bean

Cowpea mosaic virus

Foot- and-mouth disease

Foot-and-mouth disease virus VPstructural protein

[]

Cowpea mosaic virus

Mink enteritis virus

Mink enteritis virus epitope (VP)

[]

Cowpea mosaic virus

Rhinovirus

Human rhinovirus  (HRV-)and human immunodeficiency virus type (HIVepitopes)

[]

Cowpea mosaic virus Agrobacterium-mediated transformation

HIV

HIV epitope (gp)

[]

Cowpea mosaic virus

HIV

HIV epitope (gp)

[]

Cowpea mosaic virus

Mucosal vaccines not requiring adjuvants

D peptide of fibronectin binding protein B of Staphylococcus aureus

[]

Agrobacterium-mediated transformation

Autoimmune diabetes

Vibrio cholera toxin B subunit–human insulin fusion

[]

Agrobacterium-mediated transformation

Autoimmune diabetes

Glutamic acid decarboxylase

[]

Agrobacterium-mediated transformation

Cholera and E. coli diarrhea

E. coli heat-labile enterotoxin LT-B

[]

Agrobacterium-mediated transformation

Diarrhea due to Norwalk virus

Coat protein of Norwalk virus

[]

Agrobacterium-mediated transformation

Cholera

V. cholerae toxin CtoxA and CtoxB subunits

[]

Cowpea

Potato

Reference

8 Genetically modified plants: Developments and industrial aspects

161

Table 8.5 (continued ) Plant species Genetic transformation system

Disease

Vaccine antigens

Tobacco

Tmv

Cancer

c-Myc

[]

Agrobacterium-mediated transformation

Cholera and E. coli diarrhea

E. coli heat-labile enterotoxin LT-B

[]

Agrobacterium-mediated transformation

Dental caries

Streptococcus mutans surface protein SpaA

[]

Agrobacterium-mediated transformation

Diarrhea due to Norwalk virus

Coat protein of Norwalk virus

[]

Agrobacterium-mediated transformation

Hepatitis B

Recombinant HBsAg

[]

Tobacco mosaic virus

Hepatitis B

Murine hepatitis epitope

[]

Cowpea mosaic virus / Agrobacterium-mediated transformation

HIV

HIV epitope (gp)

[]

Tobacco mosaic virus

Influenza

Hemagglutinin

[]

Tobacco mosaic virus

Malaria

Malarial B-cell epitope

[]

Agrobacterium-mediated transformation

Rabies

Rabies virus glycoprotein

[]

Agrobacterium-mediated transformation

Rabies

Rabies virus glycoprotein

[]

Spinach

Reference

(i) tailoring of oil composition to be used as bio-based lubricants and biofuel in Jatropha curcas and camelina [55,112] and (ii) altered lignin composition and content to produce more efficient biofuels and other processes, including biomaterial conversion in sorghum, poplar and sugarcane [18, 34, 114].

8.4 Conclusions Genetic modifications have come a long way with diverse research applications, but the full commercial potential is yet to be envisaged. The huge industry based on raw material from plant source will achieve renovations in both the production and processing technology. The chapter has dealt with some of the modifications in different industrial arena and the genes involved in modifications, which will

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Table 8.6: Preparation of biopharmaceuticals in transgenic plants. Plants

Proteins

Genetic transformation system

Particulars

References

Human protein C (serum protease)

Agrobacteriummediated transformation

Protein C pathway

[]

Human hirudin variant 

Agrobacteriummediated transformation

Indirect thrombin inhibitors

[]

Human hirudin Oilseed, Ethiopian variant  mustard

Agrobacteriummediated transformation

Indirect thrombin inhibitors

[]

Anticoagulants Tobacco

Recombinant hormones/proteins Tobacco

Human Agrobacteriumgranulocyte– mediated macrophage colony- transformation stimulating factor

Neutropenia

[]

Human erythropoietin

Agrobacteriummediated transformation

Anemia

[]

Human epidermal growth factor

Agrobacteriummediated transformation

Wound repair/ control of cell proliferation

[]

Human hemoglobin

Agrobacteriummediated transformation

Blood substitute

[]

Human homotrimeric collagen I

Agrobacteriummediated transformation

Collagen

[]

Human serum albumin

Agrobacteriummediated transformation

Liver cirrhosis

[]

Thale cress

Human enkephalins Agrobacteriummediated transformation

Antihyperanalgesic by opiate activity

[]

Turnip

Human interferon-α

Hepatitis C and B treatment

[]

Agrobacteriummediated transformation

8 Genetically modified plants: Developments and industrial aspects

163

Table 8.6 (continued ) Plants

Proteins

Genetic transformation system

Particulars

References

Oilseed

Human enkephalins Agrobacteriummediated transformation

Antihyperanalgesic by opiate activity

[]

Rice

Human interferon-α

Agrobacteriummediated transformation

Hepatitis C and B treatment

[]

Potato

Human serum albumin

Agrobacteriummediated transformation

Liver cirrhosis

[]

Protein/peptide inhibitors Rice

Human a-antitrypsin

Particle bombardment

Cystic fibrosis, liver disease and hemorrhage

[]

Maize

Human aprotinin

Particle bombardment

Trypsin inhibitor for transplantation surgery

[]

Tobacco

Angiotensin-Iconverting enzyme

Agrobacteriummediated transformation

Hypertension

[]

Tomato

Angiotensin-Iconverting enzyme

Agrobacteriummediated transformation

Hypertension

[]

Nicotianabe thamiana

α-Trichosanthin from TMV-U subgenomic coat protein

Agrobacteriummediated transformation

HIV therapies

[]

Glucocerebrosidase Agrobacteriummediated transformation

Gaucher’s disease

[]

Daffodil phytoene synthase

Provitamin A deficiency

[]

Recombinant enzymes Tobacco

Nutraceuticals Rice

Particle bombardment

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Table 8.6 (continued ) Plants

Proteins

Genetic transformation system

Particulars

References

Potato

Amaranthus hypochondriacus

Agrobacteriummediated transformation

Amino acid deficiency

[]

Sorghum

Brown midrib gene that encodes caffeic acid O-methyltransferase, aligninproducing enzyme

–

Increase in fiber digestibility

[]

improve the efficiency cost-wise and also create wide avenues for industrial development. However, the risks involved in the application of genetic modification is still being studied and reviewed for its practical implications.

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Aman Verma

9 Chloroplast genetic engineering: Concept and industrial applications Abstract: Chloroplast engineering has emerged as an environment-friendly tool and is rather favored over nuclear engineering in some crops. Genome of plastid is highly polyploidy, and therefore it offers introduction of multiple copies of foreign gene in plant cells. Transformation of chloroplast has many inherent advantages. These advantages include higher level of foreign gene expression, multi-gene engineering in a single transformation process, absence of gene silencing and position effect variation, minimal outcrossing of transgene and precise regulation and sequestration of foreign protein. In recent years, successful chloroplast genome engineering has resulted in improved resistance to insect disease, herbicides and drought and production of biopharmaceuticals, including vaccine antigen. Furthermore, development and advancements in chloroplast transformation will help in genetic modification, genetic improvements in plants and cost-effective production of pharmaceutical components with ecofriendly approach. This chapter focuses on basic concepts of genetic engineering and its commercial prospects. Keywords: chloroplast, crop improvement, genetic transformation, therapeutic proteins, transgene

9.1 Introduction The human population is increasing continuously across the world and it is expected to be around 9.2 billion by the year 2050. In order to feed this increasing human burden on earth, food production should grow in parallel. Human largely depends on agriculture, and hence there is a concern for its growth. The major agriculture production systems aim at producing food for human utilization, fibers for fuel and feed for animals, as well as producing raw materials for other products, including colors, perfumes and drugs. Sustainable agriculture is the need of time to support both the existing and the future generations. Agriculture is said to be “sustainable” when it can continually support production at levels needed for payback (cash economy) or persistence (subsistence economy) [1]. The primary concern is that despite arable land being utilized to its maximum (High Level Expert Forum, FAO, October 2009) [2], the demands are still not met. Advances in the agricultural biotechnology arena, mainly plant genetic engineering, are believed to revitalize crop productivity. Plants have three main reservoirs of DNA: nucleus, mitochondria and chloroplast. Therefore, they are potential targets for genetic engineering. There are considerable researches pertaining to insert transgenes into the plastid genome https://doi.org/10.1515/9783110563337-009

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in spite of the nuclear genome. Chloroplast transformation has gained significant momentum as a new tool for transgene containment due to the maternal inheritance. This chapter describes recent advances in plastid transformation in agricultural sector. It laid emphasis on novel tools for plastid genome engineering, including expression of transgene to explore the potential of plastid transformation and its industrial relevance.

9.2 General features of genetic transformation Plant biotechnologists have adopted a wide range of methodology to integrate foreign DNA into plant cells. The selection of method depends upon the objectives. Each method has its own merits and demerits. The methods have been broadly classified into two categories: (a) direct DNA transfer methods; (b) indirect DNA transfer methods. The various DNA transfer methods have been listed in Table 9.1. Table 9.1: Various DNA transfer methods used for genetic transformation. S. No.

Name of the method

Merits

Demerits



Agrobacteriummediated transformation

– Most extensively used method – Has broad host range (mainly for dicotyledonous plants) – Stable integration

– Not applicable for monocotyledonous plants

[–]



Sonication-assisted Agrobacterium mediated transformation (SAAT)

– Besides transformation used as transient expression assay

– Ultrasound facilitates required

[, ]



Microparticle bombardment method (biolistic gun)

– Widely adopted for plants recalcitrant to transformation with Agrobacterium – Capable of delivering DNA into the nucleus, mitochondria and chloroplasts

– –

– Combination of Agrobacterium-mediated transformation and particle bombardment method – Co-transformation to deliver a gene of interest

–



Agrolistic transformation

–

–

References

Cell damage Inability of the transformed tissue to regenerate Expensive equipment

[–]

Expensive equipment Labor-intensive

[, ]

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Table 9.1 (continued ) S. No.

Name of the method

Merits

Demerits



Electroporation

– A large electric pulse temporarily allows polar molecules like DNA to pass into the cell – Generate transgenic plants by protoplast transformation – Used in intact plant tissues

– Cell damage by electric pulses of wrong length/ strength – Nonspecific transport during electropermeability – Ionic imbalance and cell death



Microinjection

– Micromanipulator is used – Causes release of for introduction of DNA into hydrolases and the nucleus or cytoplasm toxic compounds from the vacuole to the cytoplasm resulting in death of the protoplast

[, ]



Liposome-mediated transformation

– Microscopic spherical vesicles having DNA insert – Used for protoplast transfection

[–]



Pollen-tube pathway (PTP) method

– Foreign DNA is applied to – Low efficiency of cut styles shortly after transformation pollination – DNA reaches the ovule by flowing down the pollen tube

[–]



Silicon carbide Whisker-mediated method

– Vortex silicon carbide crystals in liquid medium containing DNA and plant cells – Physical piercing of the cell wall by crystal facilitating entry of DNA insert

[–]



Nanoparticle-mediated – Nanoparticles are method combined with chemical compounds that are difficult for cells to internalize, and these particles are then used to deliver genes into target host cells

– It is laborious and less efficient

– Low transformation efficiency

References [–]

– Lower regeneration capacity of transformants – Mainly used in animal cell transfection

[–]

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9.2.1 Chloroplast engineering Chloroplast engineering has emerged as a new tool and is rather favored over nuclear engineering in many cases. A comparative account of chloroplast engineering with respect to use of nuclear genome is shown in Table 9.2. The operon system (cluster of genes with a single region of promoter), which usually encodes enzymes and regulatory proteins of the same biosynthetic processes, is universal in prokaryotic organisms. The single transcript of whole multigene operon conveys information required for the formation of several proteins of the same pathway. In contrast, nuclear genes are transcribed singly and they are not aligned in operons. Moreover, genes of the chloroplast are often present in operons. Chloroplast engineering is also known as chloroplast transformation technology (CTT) and it is very popular in current scenario. Surprisingly, a surplus of 130 genes from different sources have been inserted, integrated and expressed successfully through CTT for different applications in important plants, including tobacco, rice, barley, maize, carrot, pine, potato, sunflower and cotton [32]. The eukaryotic plastid genomes database can be accessed at NCBI website. Researchers are also using CTT for developing resistance in crop plants against various biotic and abiotic stresses, namely, insects, pests, viral, fungal and bacterial pathogens, numerous types of herbicides, drought, cold and salt tolerance. The technology is also being used for developing phytoremediation of toxic metals, cytoplasmic male sterility, metabolic engineering, production of vaccine antigens and even for improved biofuels, biopharmaceutical agents and industrially important enzymes [33-37]. Moreover, the plastid genome transformation has many inherent values [38]. Table 9.2: Comparative advantages of chloroplast genome over nuclear genome. S. No.

Chloroplast genome

Nuclear genome



Reduced gene dispersal due to maternal inheritance

Gene dispersal is high in the environment due to its parental nature



Around  copies of a single circular chromosome per plastid

Two copies of each of many chromosomes



Approximately – chloroplasts per cell

Number of chromosomes per diploid cell is species specific



Each gene is separate and is transcribed individually

Many genes are in operons and are transcribed together



Higher expression and accumulation of foreign proteins

Lower expression and accumulation of foreign proteins



Efficient multiple gene expression in single The efficiency of single transformation for transformation event multiple gene expression is very poor

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Table 9.2 (continued ) S. No.

Chloroplast genome

Nuclear genome



A single promoter for expression of multisubunit complex protein

Several promoters for each genes to drive expression of respective subunits



Homologous recombination avoids position effects and gene silencing

Random integration presents position effects and gene silencing

These advantages (Figure 9.1) include higher levels of transgene expression, capacity to express polycistronic messages from a promoter, uniparental plastid gene inheritance to prevent pollen transmission of new DNA, absence of gene silencing and position effect variation, homologous recombination-mediated targeted gene renewal, specific transgenic control and seizing of foreign proteins in the cellular organelle to avoid its unpropitious interactions with the cytoplasmic domain [38].

Plastid transformation Multigene expression

No position effect variation

Transgene expression Chloroplast engineering

Maternal inheritance

No gene silencing

Natural containment

Figure 9.1: Advantages of chloroplast engineering.

9.2.2 Chloroplast and its genome Plastids are the site of manufacture and storage of important chemical components used by the cell. Plastids are of the following types: (1) green-colored chloroplasts containing chlorophyll; (2) yellow-, orange- or red-colored chromoplasts containing carotenoid; (3) amyloplasts storing starch granules; (4) elaioplasts containing oil; (5) proplastids (precursor of plastid found in many plant cells); (6) etioplasts (partially matured chloroplasts that are found in dark-grown seedlings). The chloroplast is the site of manufacturing of many biomolecules like sugars, starch, complex organic

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compounds and many amino acids. Reduction of nitrate to ammonium and assembling chloroplast proteins are the added functional aspects of this organelle. Plant’s chloroplast and of algal cells were evolved from photosynthetic bacteria residing inside the primitive antecedents of plants [39-41]. Chloroplast possesses multiple copies of circular genome with as many as 100–250 genes. Its genomic size varies from species to species, ranging from 107 kb of Cathaya argyrophylla to 218 kb of Pelargonium and it follows maternal inheritance in angiosperm plants [37, 42]. The copy number of genome per cell depends on the plant cell age, ranging from 1000 to 10,000. For instance, a typical photosynthetic cell consists of approximately 100 chloroplasts, and each chloroplast possesses about 100 copies of DNA. These genomes happen to be in multiple subgenomic circles and small circles are originated from larger ones through crossing over. Various genes in the chloroplast genome are aligned into polycistronic transcription units, that is, cassette of two or more genes that are transcribed by RNA polymerase enzyme from a single promoter. Epigenetic effects such as gene silencing [43, 44] and position effects [45] are not present in chloroplasts because of site-specific integration of the transgenes into the spacer regions of the genome. During CTT, gene containment occurs due to the lack of transgene transmission by pollen. During pollen maturation, plastid DNA is lost and therefore not transferred to the coming generation [46].

9.2.3 Methods for transforming plastids Plastid transformation was first accomplished in Chlamydomonas reinhardtii, a unicellular alga [47]. It was carried out for the deletion atpB gene of the chloroplast that rendered the green algae capable of carrying out photosynthesis and could be recovered by integrating the wild-type gene. The genomic DNA was coated on a tungsten microprojectile (~1 mm in size) and pushed into cells inoculated on an agar plate using a gunpowder charge [48]. Compressed gases were used for biolistic transformations of the host genome [49]. The DNA was thus incorporated into the chloroplast chromosome of this green alga by homologous recombination. Perhaps, tobacco was the first higher plant in which chloroplast transformation was successfully achieved [10, 50]. Later on, DNA was introduced into chloroplasts of the cultured cells of tobacco in situ or in leaf tissue and partial expression was observed. The transformed cells were further regenerated into whole plants with these modified chloroplasts [10, 51]. The basic layout for chloroplast engineering and its applications have been shown in Figure 9.2. Major attention of biotechnologists was driven by the experiments of McBride et al. [52]. They introduced the gene using the microprojectile bombardment method to encode the Bacillus thuringiensis lepidopteran protoxin into the chloroplast’s chromosomes of tobacco. Transgenic tobacco showed 2–3% of the soluble leaf protein expressing protoxin. Two additional approaches were used to deliver

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179

Chloroplast DNA Foreign DNA

Engineered chloroplast DNA

Therapeutic proteins

Industrial enzymes

Bio fue l

Phytoremediation

ed rov Impcrop tion duc pro

Figure 9.2: Chloroplast engineering: Basic layout and its applications.

DNA into plastids: polyethylene glycol (PEG)-mediated and direct transfer of DNA in situ. PEG-mediated approach was used in case of Nicotiana plumaginifolia [53] and Nicotiana tabacum [54] and it has the capacity to form more cells with genetically transformed plastids more easily than by the biolistic method. Knoblauch et al. [55] have shown another approach of direct injecting DNA into chloroplast genome in photosynthetically active leaf cells of tobacco. These findings mark the beginning of chloroplast engineering and opened new vistas for researchers to explore the potential of chloroplast. Chloroplast engineering demands some of the following gene features: (a) promoter region, (b) downstream box and N-terminal amino acid sequence for the codon usage, (c) 5′ untranslated region (5′ UTR) and (d) 3′ UTR. A brief description of the above cited regions is given below:

9.2.3.1 Promoter region The process of transcription in plastid is mediated by the controlled and combined actions of two types of RNA polymerases (RNA Pol) identifying distinct promoters:

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(1) T7-like single-subunit nuclear-encoded polymerase (NEP); (2) bacterium-like α2ββ′ plastid-encoded polymerase (PEP). A characteristic feature of NEP is that it catalyzes transcription in undifferentiated plastids and in certain etiolated (non-green) tissues to produce rRNAs and mRNAs, which encode for many ribosomal proteins included in the PEP also. This however leads to the amassing of active and functional PEP. Many researchers have noticed that most of the promoters of the plastids have both PEP and NEP at transcription start sites [56]. One such example is of transcription of transgenes inserted into the plastid genome using 16S rRNA promoter (Prrn16) or the psbA promoter (PpsbA). PpsbA contains transcription start sites for PEP, whereas Prrn contains transcription start sites for both PEP and NEP [57]. In another set of experiment, a synthetic promoter system had been created by changing the Prrn promoter to incorporate lac operator sequences from the Escherichia coli lac operon [58]. A T7 RNA polymerase hybrid transcription system unlike NEP has been developed to synthesize bioplastic, namely, polyhydroxybutyric acid (PHB) in the plastids [59]. Many researchers have used promoterless constructs for chloroplast engineering. A read-through transcription from the original plastid ribosomal or psbA promoters is advantageous to achieve high-level protein expression in transformed plastids [60-62].

9.2.3.2 Downstream box The region of downstream box (DB) is of 10–15 codons, present downstream of the start site. It was first recognized in E. coli [66] and found to have major effects on accumulation of foreign proteins. It functions symbiotically with the Shine–Dalgarno sequences present upstream of the start site to regulate transgene protein expression and accumulation. The last decade has witnessed many studies pertaining to the influence of DB region on the alterations of transgene protein accumulation [67, 68]. Gray et al. [61] showed that the choice of DB whether native or recombinant (fused) alters the transgene expression as well as magnitude. They reported that when 14 amino acid fusions from the N-terminal of TetC DB and NPTII DB or GFP DB were fused to either an endoglucanase gene or a β-glucosidasegene from Thermobifida fusca, the accumulation of the transgene enzymes varied almost twice the magnitude, depending upon the type of DB sequence used. For instance, TetC DB was found to be ideal for the endoglucanase gene, while NPTII DB was found to be more expressive with the β-glucosidase gene. These findings prove that choice of DB sequence should be optimized and standardized because different outcomes may be witnessed depending upon the particular coding region that is under its influence.

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9.2.3.3 5′ Untranslated regions (5′ UTRs) Since certain chloroplast genes are modulated at the posttranscriptional level [63], the integration of particular 5′ UTR into a plastid transgene may also give additional regulatory control of transgene protein expression specially designed to aim many biological processes like photosynthesis. Most regularly used 5′ UTRs are psbA gene, rbcL and the bacteriophage T7 gene 10 of the plastids. Incorporation of 5′ UTR of the bacteriophage T7gene 10 into the constructs exhibited increased expression of transgenic protein [64]. Yang et al. observed that when plants were grown in the light, the psbA 5′ UTR resulted in higher accumulation of β-glucuronidase protein encoded by uidA gene under the transcriptional control of psbA promoter. They also observed that when two additional bacteriophage 5′ UTRs along with aadA marker gene were integrated, it resulted in sufficient expression for the recovery of transgene protein [65].

9.2.3.4 3′ Untranslated regions (3′ UTRs) The 3′ UTRs are located downstream of the stop codon in the plastid genome. It consists of hairpin-loop-like structure that renders RNA maturation and posttranscriptional processing. It also checks degradation of the RNA by specific ribonucleases [69]. The 3′ UTRs are majorly derived from plastid genes, with the rbcL, rpl32, rps16 and psbA, to control foreign gene expression in plastids. Like DB sequences, 3′ UTRs should be selected carefully. In CTT, the integration of foreign genes is achieved by placing it into the intergenic spacer regions keeping in mind that the original chloroplast genes remain intact. A minimum of two flanking chloroplast genes mark the site for the integration of transgene cassettes. A transgene cassette comprises a selectable marker, such as gene for streptomycin resistance and gene(s) of interest, both under the transcriptional control of specific promoters and 3′ and 5′ untranslated regions. Knowledge of chloroplast genome sequences is vital in formulating transgene cassettes as it requires sequence information of both regulatory and flanking sequences for stable integration. Chloroplast vectors are constructed by integrating transgene cassettes into suitable bacterial plasmids, which are then bombarded into plant cells by using methods of gene transfer such as biolistic gene gun. Transgene cassettes may find its way to either nuclear or mitochondrial genome, via homologous or nonhomologous recombination events. Nontargeted integration could be easily identified by evaluation of their integration site and subsequent elimination [70]. Perhaps any nontargeted integration will not result in the transgenes expression at the nuclear or mitochondrial genome as chloroplast regulatory sequences are not functional there. Scientific community has successfully integrated many genes on chloroplasts to achieve agronomic traits of interest. For instance, transgenic tobacco has been

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engineered to combat multiple biotic and abiotic stresses with co-integration and expression of protease inhibitors and chitinase [71]. Plastid genome transformation has proved to be one of the leading options in achieving various economically important agronomic traits such as insect resistance, herbicide resistance and tolerance to drought, freezing/chilling and salt [72]. Herbicide tolerance (glyphosate tolerance) is the dominant trait that attracted the most for plastid genome transformation [51, 73, 74]. Many of such reports are summarized in Table 9.3. In addition to aiming for the improvement of agronomic traits, researchers are also focusing on the production of vaccines, therapeutic proteins and biomaterials because of the maternal inheritance of the chloroplast genes. Another important benefit associated with CTT is that one can avoid inserting antibiotic-resistant genes as selectable marker as certain chloroplast genes can act as the same. This will certainly improve public perception toward plant biotechnology and its products. One such gene is betaine aldehyde dehydrogenase (BADH) that can confer antibiotic-free selection of transformed plastids. Such an approach prevents arguments against genetically modified (GM) plants concerning about horizontal gene transfer from transgenic plants back to bacteria, which may develop antibiotic resistance.

Table 9.3: Applications of chloroplast genome engineering. Transgenes

Efficiency of expression

Engineered traits or products

Reference

Insect or pathogen tolerance Pta

.–.% Total soluble protein (TSP)

Resistance against aphid, whitefly, Lepidopteran insects, bacterial and viral pathogens

[]

Bgl-

>-fold enzyme

Resistance against aphid and whitefly

[]

RC, PG

–% TSP

Resistance to Erwinia soft rot and tobacco mosaic virus

[]

Bt cryAa operon

.% TSP

Cuboidal Bt crystals formation; % mortality of cotton bollworm, beet armyworm

[]

msi-

–% TSP

Resistance to in planta challenge of Verticillium dahlia, Pseudomonas syringae, Aspergillus flavus, Fusarium moniliforme and Colletotrichum destructivum

[]

Bt cryAa

~% of TSP

Resistance to Phthorimaea operculella

[]

cryAa

–% of TSP

Resistance to Helicoverpa zea, Heliothis virescens and Spodoptera exigua

[]

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Table 9.3 (continued ) Transgenes

Efficiency of expression

Engineered traits or products

Reference

Abiotic stress tolerance Badh

– /g Fresh weight (FW)

Salt tolerance: carrot plants survived up to  mM NaCl

[]

tps

>-fold transcript

Drought tolerance: growth in % polyethylene glycol (PEG) and rehydration after  days of drought

[]

γ-TMT

>.% TSP

Increased salt and heavy metal tolerance, enhanced accumulation of ɑ-tocopherol in seeds

[]

b-bar

>% TSP

Resistance to phosphinothricin (herbicide)

[]

EPSPS

>% TSP

Resistance to glyphosate (herbicide)

[]

panD

>-fold β-alanine

Tolerance to high-temperature stress

[]

Lycopene βcyclase

. mg/g DW

Herbicide resistance and triggers conversion of lycopene

[]

HTP, TCY, TMT

NR

Increase in vitamin E in fruit; cold-stress tolerance

[]

Other agronomic traits RbcS

>-fold RbcS transcript

Restoration of Rubisco activity in rbcS mutants

[]

TC, γ –TMT

 nmol/(h mg) FW

Vitamin E accumulation in lettuce and tobacco

[]

BicA

~.% TSP

CO capture within leaf chloroplasts

[]

Bgl-

. units Bgl g− FW

β-Glucosidase increased enzyme cocktail efficiently to release sugar from paper, wood and citrus peel

[]

ubiC

% DW

-fold higher pHBA polymer accumulation than nuclear transgenic lines

[]

Cutinase or swollenin

.% reduction of MGDG and DGDG in cutinase and .% in swollenin

Swollenin enlarged and irreversibly unwound cotton fiber; used in enzyme cocktails; cutinase showed esterase and lipase activity

[]

CV-N

~.% TSP

Increased mRNA stability and protein stability with the expression of CV-N in chloroplasts

[]

CelA,CelB

–% TSP

Hydrolyzed crystalline cellulose

[]

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Table 9.3 (continued ) Transgenes

Efficiency of expression

Engineered traits or products

Reference

bglC, celB, celA, xeg

–% TSP

Cell wall-degrading enzyme activity

[]

crtZ, crtW

>.% DW

Astaxanthin accumulation

[]

EGPh

% TSP

Chloroplast-derived β-,-endoglucanase (EGPh) was recovered from dry leaves and digested with carboxymethyl cellulose (CMC) substrate

[]

FVIII

 mg/g FW

Feeding of the HC/C antigen mixture substantially suppressed T-helper cell responses and inhibitor formation against FVIII in hemophilia A mice

[]

Proinsulin

% TSP in tobacco, % TLP in lettuce

Oral delivery of proinsulin in plant cells lowered glucose levels comparably to injectable commercial insulin

[]

IGF

.% TSP

Promoted growth of cultured HU- cells in a dose-dependent manner

[]

BACE

.% TSP

Immunogenic response against the BACE antigen in mice

[]

IFNαb

 mg/g FW

Protected cells against VSV CPE and HIV; increased MHC I antibody on splenocytes and the total number of natural killer cells and protected mice from a highly metastatic lung tumor

[]

IFN-γ

% TSP

Protection of human lung carcinoma cells against infection by encephalomyocarditis virus

[]

CTB- L

.% TSP

Immunogenic in mice following IP or oral administration

[]

LTB

.% TSP

GM ganglioside binding assay

DPT

.% TSP

[]

HEV E

. ng/μg TSP

Immunogenic in orally inoculated mice with freeze-dried chloroplast-derived multi-epitope DPT protein Immune response in mice against hepatitis E virus

p

~% TSP

Induced strong CD+ and CD+ T-cell responses in mice

[]

[]

[]

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9.2.4 Applications of chloroplast engineering 9.2.4.1 Production of vaccine antigens and biopharmaceuticals Diabetes mellitus is a chronic disease whose prevalence is continuously increasing worldwide. The World Health Organization has estimated that around 1.5 million deaths had occurred in 2012 due to this disease, making it the eighth leading cause of death. Most of the world’s population cannot afford its cure, that is, insulin, due to its high cost [37]. The production cost of insulin is high due to its requirements like fermentation systems, its expensive purification procedures from the host proteins and its costly storage and transportation. Shorter shelf-life of the finished product further adds to its cost [105, 106]. CTT can address this issue very well as it does not require such expensive production procedures and can be stored without losing the efficacy of the finished product [107, 108]. Table 9.4 has summarized many such researches aiming for engineering various vaccine antigens and biopharmaceuticals via targeting chloroplast genome of higher plants. Almaraz-Delgado et al. [109] engineered tobacco plastid for anticancer vaccine development by expressing E7 HPV type 16 protein. As already mentioned, CTT is preferred nowadays over nuclear transformation. One of the reasons behind this is the liberty for carrying out many posttranslation modifications easily such as phosphorylation, acetylation/deacetylation, amidation, formation Table 9.4: Vaccine antigens, biopharmaceuticals and therapeutic proteins engineered via chloroplast genome of higher plants. S. No.

Traits

Gene

Expression level

Reference



HIV/AIDS

gp, gp

 μg/g FW

[]



Polio virus

CTB-VP

–% TSP

[]



Tuberculosis antigens

CTB-SATCTB-MtbF

.% TSP

[]



Dengue virus

EDIII

.–. TSP

[]



Pompe disease

CTB-GAA

.–. TLP

[]



Insulin liken growth factors

IGF-n

% TSP

[]



Interferon-αb(IFN-αb)

IFN-ab

% TSP

[]



Basic fibroblast growth factor (bFGF)

bFGF

.% TSP

[]



Escherichia coli phytase gene

appA

Not detected

[]



Human glutamic acid decarboxylase

hGAD

.–.% TSP

[]

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Table 9.4 (continued ) S. No.

Traits

Gene

Expression level

Reference



Infectious burial disease virus

IBDV-VP

.–% TCP

[]



Mammary-associated serum amyloid

M-SSA

–% TSP

[]

of disulfide bonds, proper folding and the assembly of complex multimeric proteins. Another advantageous feature is the higher level of vaccine antigens or therapeutic proteins expression in green (leaves) or nongreen (fruits/roots) chloroplasts. It also provides an antibiotic-free selection system that is best suited for oral delivery of vaccine antigens against many bacterial and viral diseases like cholera, anthrax, plague, tetanus and canine parvovirus [108, 110–112]. Lakshmi et al. [113] discovered cost-effective vaccine antigens production against tuberculosis and studied their expression in chloroplasts. While working on cancer therapeutics, Tran et al. observed increased expression of foreign proteins in chloroplasts owing to its inherent capacity, which may result in toxicity on the host plant. Temporary immersion bioreactors (TIBs) using Alka Burst technology addressed this issue and produced leafy biomass that expressed OspA at levels of up to 7.6% total soluble protein to give a maximum yield of OspA. This had proved to be nontoxic for plants and particularly useful when absolute gene dispersion control is a requisite [114]. Various recombinant therapeutic proteins have been produced from a single plant C. reinhardtii. Guy’s 13 is a monoclonal antibody specific for a surface antigen of Streptococcus mutans, a bacterium that leads to dental caries. Chloroplast targets genetic construct having Guy’s 13 gene under the transcriptional control of psbA promoter. This strategy was successful in expressing IgA-G, a humanized chimeric form of Guy’s 13 antibody, with uniformly folded disulfide bonds [115].

9.2.4.2 Production of industrial enzymes and biomaterials Chloroplast genetic engineering offers best-suited solution for the synthesis of economically and industrially important biomaterials such as amino acids, enzymes and technical proteins for multiple purposes. In one such instance, transgenic tomatoes were engineered to express milk proteins, human β casein, through CTT. Another important aspect is to produce biodegradable plastics that can be a miraculous alternative to synthetic plastics. Direct conversion of chorismate to p-hydroxybenzoic acid (pHBA) was achieved by integrating gene for chorismate pyruvate lyase (CPL), an enzyme encoded by the ubiC gene, in E. coli. A 10–20-fold increase in pHBA production in tobacco’s nuclear transformation was

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noticed, which is far more than the required quantity for a commercially viable pathway [89]. However, the highest level of the poly (pHBA) polymer (25% dry weight) in normal healthy plants was optimized in chloroplast genomes [125]. An operon extension strategy was used for the production of the renewable biodegradable plastic polyhydroxy butyrate (PHB) in tobacco (N. tabacum) [126]. A lot of inputs have been witnessed to produce PHB through different systems, but till date maximum level of PHB was achieved in plastids only. This was due to the high substrate flux of the PHB pathway, that is, acetyl-CoA, during fatty acid biosynthesis in the plastids [127]. Xylanase is an important cellulolytic enzyme and biomaterial that finds its utility in the fiber, paper, animal feed and brewing industries. The first of nuclear transformation was unsuccessful in plants expressing xylanases due to cell wall degradation. However, 6% tsp xylanase accumulation was achieved when the xynA gene was integrated into the tobacco chloroplast genome [128]. Certain enzymes of amino acid biosynthesis are also targeted through CTT. One of the enzymes of tryptophan (Trp) synthesis and responsible for its regulation is anthranilate synthase (AS). Scientists engineered transgenic plants showing high levels of ASA2 mRNA. Higher expression of the AS α-subunit and a fourfold increase in AS enzyme activity were achieved, which was also found to be less sensitive to feedback inhibition of Trp biosynthesis. Resultant plants exhibited sevenfold increase in free Trp in the leaves of transgenic plant [129]. This finding has also opened new vistas of using ASA2 gene of tobacco as a selectable marker. Economically important examples of enzymes and biomaterials that have been processed through chloroplast engineering within the tobacco genome are mentioned in the Table 9.5. Table 9.5: Biomaterials and enzymes engineered via chloroplast genome of tobacco. S. No.

Biomaterials/enzymes

Gene

Expression level

     

Polyhydroxybutyrate Xylanase β-Glucosidase Superoxide dismutase Cellulases endo-Glucanase

phb operon xynA Bgl Cu/ZnSOD bglC, celB, celA,xeg celB

.% TSP % TSP  mg/g TSP % TSP –% TSP –% TSP

Reference [] [] [] [] [] []

9.2.4.3 Phytoremediation Phytoremediation is a key solution to increasing threat of environmental pollution due to increasing urbanization and land uses. It is a safe and economic way for cleaning up environmental contaminants using plant system. Although plants

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possess limited potential to eradicate high levels of toxic chemical agents, their remediation capacity can be enhanced through genetic engineering aiming especially chloroplast genomes of plant. The most hazardous are the organomercurial compounds that mainly target chloroplast in plants. It is one of the most toxic forms of mercury. It is, thus, necessary to engineer chloroplast genome to provide added resistance and potentiate its detoxification ability for organomercurial compounds and metallic mercury [124,132]. Synthesis of metal chelators via CTT can do wonders in this issue. This will improve the capability of plants for metal uptake [7, 133, 134] and its sequestration. In this direction, Occhialini et al. integrated two bacterial genes encoding, mercuric ion reductase (merA) and organomercurial lyase (merB), as an operon in the chloroplasts of tobacco. Resultant transgenic plants accumulated mercury primarily in roots, reducing its concentration in soil, up to 200 μg/g. Furthermore, they are found to accumulate 100-fold more of mercury in the leaves than the control untransformed plants without showing any detrimental effect [135]. Phytoremediation of toxic mercury was also achieved by integrating metallothione in tobacco’s chloroplast via CTT [136].

9.2.4.4 Production of biofuels CTT can also be explored in terms of production of biofuels. The foremost requisite for producing lingocellulose-based biofuels is to develop a rapid and cost-effective enzymatic system for speedy biomass depolymerization. Chloroplast acts as an ideal bioreactor for large-scale enzyme production due to its increased levels of protein expression and compartmentalization of toxic proteins within chloroplasts of transgenic plants [137]. Production of chloroplast-originated mixture of enzymes for production of fermentable sugars from various lignocellulosic biomass marked the stepping-stone in the area of biofuels. Biofuel-based researches are flooded with many examples of enzymes from various sources like bacteria, fungi and moulds, which are integrated into tobacco chloroplasts for the production of fermentable sugars. Few such examples are β-glucosidase, β-1, 4-endoglucanase, swollenin, cutinase, endoglucanases, esterase, exoglucanase, pectate lyases, acetyl xylan esterase, xylanase, xylan and lipase [61, 95, 138–140]. CTT-derived cutinase, due to its expansion activity, showed enlarged segments and easier unwinding of the intertwined inner fiber of cotton. It also exhibited esterase and lipase activity [95]. Another instance of β-mannanase enzyme isolated from Trichoderma reesei showed six- to sevenfold higher enzyme activity than E. coli. Pantaleoni et al. showed that when chloroplastderived β-mannanase enzymatic mixture was used, 20% more glucose equivalents were produced from pinewood than the controls without mannanase [139]. Many other chloroplast-based enzymes like cutinase and pectate lyase A from Fusarium solani; endoglucanase exoglucanase from Clostridium thermocellum; endoglucanase, swollenin, acetyl xylan esterase and xylanase enzymes from T. reesei and lipase from

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Mycobacterium tuberculosis showed broader pH and higher temperature stability than enzymes expressed in E. coli. In one of the studies, 36-fold higher glucose was produced from citrus peel; filter paper/pine wood when treated with chloroplast derived crude extract enzymatic cocktails as compared to the commercially available one [41].

9.2.4.5 Conferring stress tolerance As already mentioned, chloroplast is one of the best-suited sites for the improvement of many agronomic traits due to its higher level of gene expression and subsequent containment via maternal inheritance. Recently, conferring stress tolerance research area is gaining momentum. Many scientific groups are constantly working on targeting this miraculous organelle with special focus on herbicide resistance, insect resistance and tolerance to other biotic and abiotic stresses. Herbicide resistance Herbicide is the most studied and experimented area of the biotic stresses. Normally, transgenic plants developed for this trait are mostly insensitive to the hazardous effects of herbicides. Glyphosate is a broad-spectrum and most popular herbicide whose function is to inhibit the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSPS) of shikimic acid pathway forming aromatic amino acids like tyrosine. It is encoded by a nuclear genome but is expressed within the chloroplast. Therefore, CTT plays a vital role in achieving glyphosate resistance [72, 73, 141]. The first successful report was of Daniell et al. [51]. They were able to express chloroplast-targeted eukaryotic gene in petunia. The aroA gene was inserted either between trnI and trnA genes in the inverted repeat region or between the rbcL and accD genes in the large single copy region [51]. This stable integration was able to increase resistance even more than the lethal concentration. Another experiment showed the incorporation of CP4 EPSPS gene in the plastids of tobacco, which provided around 250 times more EPSPS enzyme in comparison to the nuclear transgenic experimentation [67]. The second most common herbicide used worldwide is phosphinothricin, commonly available as Basta/Bialaphos. Chloroplast engineering in tobacco plants by bar genes showed field-level tolerance when compared with the controls [82]. Insect resistance Worldwide setbacks are incurred due to insect attack directly leading to production losses. Most of the strategies have aligned for insecticidal protoxins integration in plants produced by B. thuringiensis. The Cry2Aa2 is an operon, possessing target genes Cry2Aa2, orf 1 and orf 2. De Cosa et al. [43] recorded highest level of the chaperone activity of orf 2 and were able to express Cry2Aa2 protein up to 46% tsp in tobacco chloroplasts. The expressed foreign gene provided protection from the

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degradation by overexpressing chaperone crystal protein of B. thuringiensis. Reduced level of the same via the nuclear genome transformation poses the threat of developing Bt-resistant pests. In contrast, CTT-derived plants were able to cause damage to the insects that could bear insecticidal protein concentrations as high as 40,000 times than normal, even at low levels of Cry2Aa2 gene expression [79]. In another set of experiment, Cry1Ia5 insecticidal protein was found to be accumulating up to the levels of 3% of tsp in tobacco leaf tissue by using protoplast transformation [142]. The antimicrobial peptide MSI-99 (an analogue of maganin-2) confers protection by targeting negatively charged phospholipids, which are especially found in the outer membranes of bacteria and fungi, against many prokaryotic organisms [143]. Additionally, fungal pathogen like Colletotrichum also showed similar responses by using chloroplast engineering. The case study of MSI-99 in tobacco clearly indicated the contrasting features of chloroplast genome engineering over nuclear genome in conferring adequate disease resistance against pathogens. CTT was sufficient enough in providing protection from both bacterial and fungal pathogens [144].

9.6.5.3 Osmotic stress resistance Environmental stresses such as salinity, freezing and drought can affect extremely leading to osmotic imbalance and in turn cellular dehydration that restricts plant growth and development. Osmoprotectants are the choice of molecules whose function is to maintain osmotic balance and help the organism to survive under stressed conditions [145]. Solutes such as sugars, betaine and amino acids are produced in lieu of stressed condition. Their role is to support turgor pressure and maintain the integrity of cellular macromolecules and biological membranes from damage due to high salt concentrations. Most of the work in this direction is on trehalose, an osmoprotectant synthesized by trehalose phosphate synthase enzyme encoded by the TPS1 gene. Under stress conditions of heat, freezing, drought and salt, it accumulates and protects the plants from the deleterious effects. CTT-derived transgenics showed normal growth and were able to accumulate trehalose as much as 25 times higher [44]. Drought tolerance was analyzed by keeping CTT-derived transgenic plants in 6% polyethylene glycol (PEG) normal and healthy growth was observed in comparison to controls that were fairly bleached. The aforementioned threat of stress increases salinity in the soil, which is also due to excessive use of fertilizers and rainfed mode of irrigation in the present day. This not only reduces metabolite exchange but affects photosynthetic capacity in plants as well. Increasing the production of glycine betaine (GB) is found to be highly effective in many flowering plants and marine algae under such conditions. It is a quaternary ammonium osmolyte and hydrophilic in nature. However, many important cash crops do not synthesize or do not accumulate sufficient levels of GB. An experiment targeting the nuclear

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genome of spinach with badh (betaine aldehyde dehydrogenase) gene showed salt tolerance to a lesser extent. However, when carrot plastids were promoted to overexpress badh gene, a significant amount of tolerance was conferred in resultants due to the accumulation of GB against salt stress [80]. Furthermore, choline oxidase enzyme isolated from bacteria Arthrobacter globiformis was able to form GB in a single enzymatic reaction in spite of two in the former case. Oryza sativa L., which does not synthesize GB at all, when transformed with coda gene from A. globiformis via CTT showed stress tolerance to a greater extent. Another active osmolyte is an amino acid, that is, β-alanine. Increased expression of this in plants showed promising results in enhancing environmental stress tolerance. Decarboxylation of aspartate by enzyme l-aspartate-alpha-decarboxylase (AspDC) to form β-alanine and carbon dioxide in E. coli is under the control of panD gene encoding for AspDC. A constitutive E. coli panD expression cassette was coinserted into the chloroplast genome of tobacco with a constitutive aadA gene as a selectable marker gene through biolistic gene gun. This results in the accumulation of β-alanine in transplastomic plants four times higher than nontransgenics. The resultants also showed 30–40% increased biomass due to CO2 assimilation in transplastomic plants under heat stress [83].

9.2.4.6 Enhancing nutrition Vitamin E-enriched oils are obtained from the seeds of rapeseed, soybean, groundnut and maize. The most abundant form of vitamin E is γ-tocopherol, but its biological active form is marked by α-tocopherol, which is relatively low in content. However, sunflower (Helianthus annuus) seed oil is quite rich in α-tocopherol [146]. α-Tocopherol is synthesized from its immediate precursor γ-tocopherol with the help of enzyme γ-tocopherol methyl transferase (γ-TMT). This also represents the rate-limiting step of the pathway [147]. Integration of the γ-tmt gene into the chloroplast genome of the seeds showed overexpression of γ-TMT in the inner layer of chloroplast. The conversion rate of γ-tocopherol to α-tocopherol was found to be approximately 10 times higher in seeds [81]. Synthesis of provitamin A, that is, β-carotene, is another example where lycopene β-cyclase (lcy) genes is integrated into the tomato plastids. This has enhanced conversion of lycopene into β-carotene many folds [148].

9.2.4.7 Engineering nitrogen fixation Nitrogen fixation is a costly affair in terms of energy utilization, that is, ATP consumption, and chloroplasts play a major role in bearing this energetic load through photosynthetic energy transduction products as a source for the same [149, 150].

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Nitrogenase reductase enzyme is sensitive to the presence of O2, which is the major by-product of photosynthesis and thus appears to restrict N2 fixation by chloroplasts. However, cyanobacteria perform the nitrogen fixing process simultaneous to oxygenic photosynthesis. These two contrasting but important processes are possible by applying division of labor rule. Cyanobacteria segregate these two processes where thick-walled heterocyst fixes the atmospheric nitrogen as they are potent enough in blocking the entry of inhibitory oxygen. Other than this, heterocysts photosynthetic apparatus lacks PSII apparatus that produces oxygen as a product but at the same time contained PSI to proceed for cyclic photophosphorylation to gain ATP.

9.2.4.8 Engineering of photosynthesis Photosynthesis probes either by using chloroplast gene deletion or by mutagenesis. Bock and Khan [151], along with other groups, have tried to determine which ORFs of the chloroplast are vital for photosynthesis. Rubisco is the foremost choice of the scientists as a candidate for genetic engineering because of its potential for efficient atmospheric carbon fixation in vascular plants [152], primarily in C3 plants that lag behind C4 plants in terms of carbon-concentrating mechanisms [153, 154]. Gene replacement approaches have been explored to identify the factors that are responsible for the functioning of Rubisco like its preferences for CO2 versus O2 [155]. Kanevski and Maliga [156] found that functional Rubisco is formed when its nuclear-encoded large subunit is synthesized in the cytoplasm and with the aid of chloroplast transit peptide (CTP) is imported into chloroplasts. Fused C-terminus of the tobacco rbcL gene with a 6×Histag could also ascertain origin of functional Rubisco [157]. Transformation of heterologous Rubisco subunits rbcL from tomato of the same family into the chloroplast genome of tobacco revealed mixed results (Table 9.6). Hybrid enzymes with tobacco and tomato larger subunits produced pale/yellow-green plants that could grow photoautotrophically without the need of excess of CO2 [158]. When the tobacco’s larger subunit was exchanged with that of Synechococcus PCC6301, pale-yellow-colored plants were developed that needed higher concentration of sucrose for growth [159]. Results pertaining to the concentration of Rubisco needed for optimal growth and development of plants resulted in varied conclusions because of its sluggish nature. This is the reason that most of the part of stroma is filled with the same enzyme. Similar reason was proved in case of wheat [160]. However, results were contrasting in case of rice where addition of rbcS genes could not show much improvement in the photosynthesis [161]. In spite of this many plants showed normal phenotypes in greenhouse and in growth chambers under lower Rubisco levels. In another study, a thermostable Rubisco activase mutant was identified whose presence showed improved photosynthesis and growth of Arabidopsis [162], but to a limited extent as overexpression of the same was not beneficial in rice [163].

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Table 9.6: Photosynthesis improvement through chloroplast transgenic expression and increased expression of various enzymes. S. No.

Type of gene alterations

References



Deletion of rbcL from plastome

[]



Addition of ×His onto rbcL in plastome

[]



Replace tobacco rbcL with tomato rbcL

[]



Replace tobacco rbcL with rbcM from Rhodospirillum rubrum

[]



Replace tobacco rbcL with rbcL from C Flaveria

[]



Replace tobacco rbcL with gene encoding-linked Synechococcus large and small subunits

[]



Enzyme improvement – Sedopheptalose-,-bisophosphatase (SBPase) – Fructose-bisphosphate aldolase (FBP aldolase) – Bicarbonate transporters – Rubisco activase – Bacterial glycolate to glycerate pathway (five proteins) – Glycolate oxidase (GO), malate synthase (MS) [] – Cyanobacterial ictB gene []

[, ] [, ] [] [] [] []

This has also been revealed that if one alters the concentration of enzymes of photosynthetic carbon assimilation and metabolism, expression of enzymes like SBPase would be increased and could possibly accelerate the rate of light-saturated photosynthetic reactions (Table 9.6). Examples of such candidate enzymes on which scientists have put in efforts are fructose-1, 6-bisphosphate aldolase (FBP aldolase), UDP-glucose phosphorylase and ADP-glucose pyrophosphorylase (ADPGPP) [174]. People have worked on improving photosynthetic efficiency by attempting to lower down the extent of photorespiration through CTT [162]. One major breakthrough was to get a C3 plant to behave like a C4 by introducing C4 photosynthetic machinery into C3 plants like famous C4 rice project [175, 176].

9.3 Limitations to plastid transformation technology One of the leading constraints limiting CTT potential is the present inability to derive regenerated homoplasmic monocotyledonous transplastomic plants, especially in case of promoting photosynthesis in crop plants [177]. The case of monocotyledonous

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grasses demands a whole new range of selectable markers and plant tissue culture techniques to get the stable transformants. Possible choices feasible for CTT are flooded in many review articles [178, 179]. Major effort of the hour is to make nuclear transformation feasible for major crop plants where CTT is not finding its way. It is, however, necessary to widen up applications of CTT for a number of important plants.

9.4 Conclusion and future prospects Enormous advantages are listed here of chloroplast genome over nuclear genome as far as plant genetic transformation efforts are concerned. The nuclear genome targeting strategies are not able to deliver products when a higher rate of expression and multigene engineering (more than one gene transfer engineering) are a prerequisite. Chloroplast transformation offers a wide range of unique advantages over other biotechnological applications as discussed in this chapter: improving photosynthetic efficacy, conferring agronomic traits, producing industrial important enzymes, biofuels, biomaterials, phytoremediation, molecular farming for antibiotics, biopharmaceuticals and vaccines. Many crops acted as a candidate for CTT such as tomato, tobacco, Arabidopsis, carrot, lettuce, potato, oilseed rape, sugar beet, cotton, petunia, cabbage, soybean, rice, poplar eggplant and sugarcane [180]. Many successful attempts have been made in CTT, but there is still long way ahead that might be more challenging than earlier cases as with the monocot grasses. Challenges are like recalcitrant nature of cereal crops as mentioned above to the available regeneration protocols. Obtaining an efficient stable shoot regeneration system is the key to success in these crops [181]. Second, standardization of huge level of expression foreign proteins is to be under control as the phenotypic alterations of transplastomic plants can lead to severe losses [182]. Other challenges are to tackle the lack of appropriate tissue-specific regulatory and promoter sequences [183, 184] and the case gene expression in nongreen plastids [185]. Efficient recovery of the desired transplastomic transformants [186] and degradation of foreign proteins includes other such hindrances to the CTT [187]. Moreover, CTT provides marker-free transplastomic plants that will certainly ease public concern and boost up the public acceptance for this miraculous organelle. Despite many constraints, chloroplast genome targeting is still an attractive site and gaining momentum for wonders to come, which will leave huge impact on humans and rest of the living world.

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[98] Youm JW, Jeon JH, Kim H, Min SR, Kim MS, Joung H, et al. High-level expression of a human beta-site APP cleaving enzyme in transgenic tobacco chloroplasts and its immunogenicity in mice. Transgenic Res. 2010, 19, 1099–108. [99] Arlen PA, Falconer R, Cherukumilli S, Cole A, Cole AM, Oishi KK, Daniell H. Field production and functional evaluation of chloroplast-derived interferon-α2b Plant Biotechnol J 2007, 5, 511–25. [100] Leelavathi S, Reddy VS. Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors. Mol Breeding 2003, 11, 49–58. [101] Molina A, Hervas-Stubbs S, Daniell H, Mingo-Castel AM, Veramendi J. High yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnol J 2004, 2, 141–53. [102] Soria-Guerra RE, Alpuche-Solis AG, Rosales-Mendoza S, Moreno-Fierros L, Bendik EM, Martinez-Gonzalez L, Korban SS. Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer. Planta 2009, 229, 1293–302. [103] Zhou YX, Lee MYT, Ng JMH, Chye ML, Yip WK, Zee SY, Lam E. A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice. World J Gastroenterol. 2006, 12, 306–12. [104] Gonzalez-Rabade N, McGowan EG, Zhou F, McCabe MS, Bock R, Dix PJ, et al. Immunogenicity of chloroplast-derived HIV-1 p24 and a p24-Nef fusion protein following subcutaneous and oral administration in mice. Plant Biotechnol J 2011, 9, 629–38. [105] Farran I, McCarthy-S I, Río-M F, Mansilla C, Lasarte JJ, Mingo-CM. The vaccine adjuvant extra domain A from fibronectin retains its proinflammatory properties when expressed in tobacco chloroplasts. Planta 2010, 231, 977–90. [106] Spök A, Karner S, Stein AJ, Rodríguez-C E. Plant molecular farming, opportunities and challenges. JRC Scientific and Tech Rep. 2008, 1–146. [107] Holtz BR, Berquist BR, Bennett LD, Kommineni VJ, Munigunti RK, White EL. Commercial-scale biotherapeutics manufacturing facility for plant made pharmaceuticals. Plant Biotechnol J 2015, 13, 1180–90. [108] Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, Alpuche-Solís AG, Martínez-González L, Korban SS. Expression of an immunogenic F1-V fusion protein in lettuce as a plant-based vaccine against plague. Planta 2010, 232, 409–16. [109] Almaraz-Delgado AL, Flores-Uribe J, Pérez-España VH, Salgado-Manjarrez E, Badillo-Corona JA. Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii. AMB Express 2014, 4, 57. [110] Daniell H, Chebolu S, Kumar S, Singleton M, Falconer R. Chloroplast derived vaccine antigens and other therapeutic proteins. Vaccine 2005, 23, 1779–83. [111] Davoodi-Semiromi A, Schreiber M, Nalapalli S, Verma D, Singh ND, Banks RK, et al. Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery. Plant Biotechnol J 2010, 8(2),223–42. [112] Michoux F, Ahmad N, Hennig A, Nixon PJ, Warzecha H. Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes. Planta 2013, 237, 903–8. [113] Lakshmi PS, Verma D, Yang X, Lloyd B, Daniell H. Low cost tuberculosis vaccine antigens in capsules: expression in chloroplasts, bioencapsulation, stability and functional evaluation in vitro. PLoS ONE 2013, 8, e54708. [114] Tran M, Van C, Barrera DJ, Pettersson PL, Peinado CD, Bui J, et al. Production of unique immunotoxin cancer therapeutics in algal chloroplasts. Proc Natl Acad Sci US 2013,110, E15–22.

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Ram Kumar Pundir, Pranay Jain, Satish Kumar, Mukesh Yadav, Rajesh Kumar

10 Plant biotechnology: Industrial prospects and scopes Abstract: Plant biotechnology involves use of multiple techniques in a systematic and productive manner to accomplish the needs and objectives of humans along with associated lives. Plant biotechnology covers nearly all aspects, from molecular to product level, for production of value-added ecofriendly industrial products in a cost-effective manner for human welfare. Plant biotechnology as a subject has very broad industrial future perspectives for human welfare. Plants have a tremendous potential to produce metabolites, enzymes, medicines and edible vaccines and remediate the unwanted substances using recombinant DNA technology. Plants-derived vaccines, medicines and secondary metabolites are of immense industrial potential. Plants secondary metabolites are of particular interest in different industries, including food and pharmaceuticals. In the current scenario, the world population is more inclined toward the plant products as compared to their pure chemical alternatives. This chapter focuses on industrial applications and future prospective of plant biotechnology. Keywords: commercial aspects, edible vaccines, enzymes, metabolites, phytomedicine, phytoremediation, plant biotechnology, value addition

10.1 Introduction Plant biotechnology is an important tool of modern science to satisfy the human and associated lives. Plant biotechnology may be defined as generation of useful products and services from plant cells, tissues and various other parts of plants. Plant biotechnology can feed the increasing population and also support the other commercial requirements of humans at global level. Plant tissue culture and plant genetic engineering are among the most important applied aspects of plant biotechnology. Micropropagation involves the practice of rapidly multiplying stock plant material to produce a large number of progeny plants with the help of modern plant tissue culture methods. Micropropagation is used to multiply plants that have been genetically modified or bred through conventional plant breeding methods. Various plant-based products, including secondary metabolites, carotenoids, pigments, oils, resins, flowers, fruits, seeds, medicines, edible vaccines and other components, are continuously in demand. Some examples of plant metabolites and plant-derived products are listed in Table 10.1. https://doi.org/10.1515/9783110563337-010

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Table 10.1: Some examples of plants having potential to be explored at industrial level for various purposes. Category of product

Compounds/plant sources

Properties

Flavonoids

– Anthocyanins – Chalcone – Flavanones – Flavones – Flavonols – Isoflavonoid

– Antioxidant – Anti-inflammatory – Anticarcinogenic – Antimicrobial – Antimutagenic – Potent inhibitor of some enzymes

Phytomedicine

– Allium sativum – Plumbago indica – Semecarpus anacardium – Hemidesmus indicus – Terminalia arjuna – Withania somnifera – Ocimum sanctum

– Cardiovascular disease

Phytomedicine/herbal formulation

– Emblica officinalis – Terminalia chebula – Terminalia bellirica

– Arthritic disease

Phytomedicine/herbal formulation

– Echinacea purpurea – Echinacea pallida

– Respiratory tract disease

Phytomedicine/herbal formulation

– E. officinalis – T. chebula – T. belarica

– Digestive system disease/disorders

Plant pigment as bio-color

– Chlorophyll – Betacyanins – Flavonoids – Anthocyanins – Carotenoids

– Green-yellow color – red-orange-yellow, bluishred, red-violet – Red, purple – Blue – Yellow-orange-red

Phytoremediation

– Cynodon dactylon – Paspalum notatum – Populus nigra – Brassica juncea – Brassica campestris – Thlaspi caerulescens – Arabidopsis sp.

– Heavy metals – Pollutants

Secondary metabolites

– Terpenes and steroids – Phenolic compounds – Polyketide and fatty acids – Phenylpropanoids – Alkaloids – Flavonoids

– Health benefits – Perfume industry – Cosmetics – Food supplements – Bio-colors

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Using scientific tools and techniques, expressions of plant molecules, metabolites and traits can be controlled to make a desired level of changes. Moreover, with the help of plant biotechnology, the nutritional value of raw food can be increased by altering the specific metabolic pathway. Scientists are extensively working one expanding the commercial dimensions of plant biotechnology and the related fields. This chapter focuses on major plant-derived commercial aspects or value-added products of prime importance.

10.2 Plant secondary metabolites and phytomedicines A large number of structurally diverse secondary metabolites occur in plants and are stored in higher concentrations, sometimes in organs that generally do not produce them. Plants have evolved secondary metabolites with a broad spectrum of biochemical and pharmacological properties. Currently, many plant secondary metabolites are well known for their diverse biological activities. Broadly, these activities include antibacterial, antifungal and anticancer activities. Around 20,000 plant species are used in traditional medicines and they are prospective reservoirs for new pharmaceuticals. In recent years, traditional medicinal plants have received huge attention as their phytochemicals may lead to new drug discoveries [1]. It is now well established that different plants and their various parts possess different phytocompounds.

10.2.1 Plant secondary metabolites Recently, focus has been on utilizing ecofriendly and biological safe plantderived products of industrial demand or repute. The approach may be to increase nutrients or to prevent and treat the various diseases. Plants produce thousands of different types of chemicals, including pharmaceutical components, aromatic compounds, pigments, cosmetics, nutraceuticals and other chemicals of industrial importance (Figure 10.1). Plant cells carry out both primary and secondary metabolism [2]. Primary metabolism involves the synthesis of core polymers, especially polysaccharides, proteins, lipids, nucleic acids and amino acids. Secondary metabolism is particularly active at specific stages of growth and development. The environmental factors and microbial interactions may also be responsible for inducing the secondary metabolism in plants at a specific stage or season. Generally, secondary metabolites are derived from primary metabolites through chemical modifications like methylation, hydroxylation and glycosidation. Therefore, secondary metabolites are more complex in

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Figure 10.1: Industrial aspects of plant biotechnology.

comparison to primary metabolites [2, 3]. Secondary metabolites are classified on the basis of their chemical structure (aromatic rings and sugars), composition (presence or absence of nitrogen content) and solubility in various solvents. Some secondary metabolites are classified according to the pathways by which they are synthesized. Some of the basic and important secondary metabolites are categorized as alkaloids, phenolics, glycosides, flavonones, flavonoids and flovonols, terpenes and saponins.

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10.2.2 Phytomedicines Phytomedicines are plant-based therapeutic molecules or formulations having various active components. Nature is one of the most powerful and primitive sources of drug that include plants, animals and microbes and mineral sources. The drugs derived from the Nature are called natural products. Natural products, especially phytochemicals, are useful in search of novel chemicals entities. Scientifically, the routine procedure involves various steps to develop a commercial formulation or prepare the phytomedicine. These steps are to be followed sequentially to successfully employ the phytomedicines in market. The various steps involved are as follows: 1. Collection of plant material 2. Drying and powder formation 3. Dissolve in solvents for extraction 4. Filtration to get plant filtrate/extract 5. Screening for bioactivity 6. Phytochemical analysis 7. Purification of potent phytochemicals 8. Characterization of most effective bioactive phytochemical agent 9. In vivo studies of most effective bioactive phytochemical agent 10. Final clinical trials and FDA approval 11. Packaging and selling The phytomedicines have some oblivious advantages that make them molecules of interest in the modern era of drug design development. The natural products are still contributing a major part in the form of starting material for drug discovery. A large number of new chemical entities are either natural derivatives or semisynthetic derivatives of natural products. The plant-based metabolites have been shown to have antimicrobial, antidiabetic, anticancer, antihypertensive and antiinflammatory properties.

10.2.2.1 Phytomedicines for cardiovascular diseases Cardiovascular diseases (CVDs) are among the leading causes of death globally [4, 5]. Herbal medicine contains various biologically active natural products. A number of herbal products have been employed at global level for the management of CVD [5, 6]. Moreover, this culture has been passed on to the modern generation [5, 6]. Intake of certain flavonoid classes reduces cardiovascular disease risk [7]. Some experts have recommended increased intake of flavonoid-rich diets as preventive measure [7]. Also, consumption of plant foods is associated with lower risk of CVD [8, 9].Flavonoids are bioactive noncaloric, polyphenolic and non-nutrient secondary metabolites in plants. Flavonoids have potent antioxidant, anti-inflammatory activity, reduce LDL cholesterol

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oxidation, regulate endothelial nitric oxide synthesis and some also have weak estrogenic activity [10]. Phenolics are derivatives of benzene (cyclic derivatives in the case of polyphenols) with one or more hydroxyl groups associated with ring structure. They are classified into 10 different groups, depending upon their chemical structure. The two main types of polyphenols are flavonoids and phenolic acids. Flavonoids are distributed as flavones, flavanones, isoflavones, flavonols, flavanols, proanthocyanidins and anthocyanins. The common flavonoids are quercetin (a flavonolpresent in onion, tea and apple), catechin (a flavanolpresentin tea and several fruits), hesperetin (a flavanone present in citrus fruits), pigment anthocyanin (red fruits; tomatoes, blackcurrant, watermelon, raspberry, pomegranate, strawberry), daidzein (isoflavone in soybean) and proanthocyanidins (commonly present in apple, grape and cocoa). Guggul (Commiphora wightii), Garlic (Allium sativum), Arjuna (Terminalia arjuna) and Hawthorn (Crataegus oxyacantha) have been reported for their cardiovascular effects. These plants are known for use in the treatment of heart disease for hundreds of years and can be utilized effectively in the treatment of cardiovascular diseases, including ischemic heart disease, congestive heart failure, arrhythmias and hypertension [11].

10.2.2.2 Phytomedicines as antioxidants Oxidative stress has been identified as the major reason behind the development and progression of numerous diseases [12]. Plants have an innate ability to synthesize a wide range of antioxidants (nonenzymatic) capable of attenuating reactive oxygen species (ROS)-induced oxidative damage. Although monoterpenes, sesquiterpenes, diterpenes and other alkaloids have shown antioxidant activity, phenolic antioxidants appear to be the most important due to their promising antioxidant activity under both in vivo and in vitro conditions. Plant phenolics are mainly classified into five major groups: phenolic acids, flavonoids, lignans, stilbenes and tannins [12–15]. Flavonoids and phenolic acids have been found to have excellent antioxidant activity in both in vitro and in vivo studies. They are also known to interact with other physiological antioxidants, including ascorbate or tocopherol, and to synergistically amplify their biological effects [16]. It can be concluded that phytochemicals play a vital role in preventing oxidative damage. At cellular and molecular levels, they inactivate ROS. Natural antioxidant phytochemicals in food are of great interest due to their beneficial effects on human health. These antioxidants offer protection against oxidative deterioration [17].

10.2.2.3 Phytomedicines against diabetes Diabetes mellitus is a complex metabolic disorder resulting from insulin insufficiency or insulin dysfunction. Type I diabetes (insulin-dependent) is caused due to

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insulin insufficiency. Type II diabetes (insulin-independent) is due to immunological destruction of pancreatic β-cells that results in insulin deficiency. Type II diabetes is characterized with insulin resistance. It is the more common form of diabetes constituting major part of the diabetic population [18]. Since ancient times, traditional herbal medicines are in use all over the world. The side effects of insulin therapy and other oral hypoglycemic agents impose the use of more effective and safer antidiabetic drugs. Several medicinal plants have shown antidiabetic activity in experimental and clinical investigations. Plants such as Momordica charantia, Mucuna pruriens, Murraya koenigii, Eugenia jambolana and Brassica juncea have been reported to have antidiabetic properties [19]. In the recent years, hundreds of plant-derived medicines have been reported to cure and treat diabetic. Major attention has been given to Vernonia amygdalina, Morinda lucida, Carica papaya, Citrus aurantiifolia, Bidens pilosa, Ocimum gratissimum and M. charantia. Besides these herbal candidates, aloe vera has also been reported to have beneficial effect in treatment of diabetes [20]. The clinical trials of aloe vera have resulted in positive results. The oral administration of aloe vera juice resulted in reduced blood sugar and triglyceride levels in the treated patients. The results suggested the potential of aloe vera juice for use as an antidiabetic agent. The positive effects of aloe vera are due to the presence of compounds such as polysaccharides, mannans, lectins and anthraquinines [21].

10.2.3 Plant resources as nutraceuticals As already mentioned, plants have been used as food and medicine since ancient times and they are important part of our routine diet [22]. The term “nutraceutical” may be defined as food or part of food that provides medical or health benefits, including the prevention and/or treatment of diseases [23, 24]. It can also be defined as diet supplement that delivers a concentrated form of a biologically active component of food in a nonfood matrix to enhance health [25, 26]. It also refers to natural functional or medical food or bioactive phytochemicals that have health-promoting potential and also disease-preventing medicinal properties. Nowadays, a large group of consumers recognize the relationship between nutrition and health, in relation to “functional foods” and “nutraceuticals” [22]. Therefore, the demand for functional food and nutraceuticals is growing rapidly and the associated market is growing sharply. Consumers are now aware of diet-related health problems and need to increase consumption of fruits and vegetables in daily routine. According to FAO [27], phytochemicals and their metabolic products may inhibit pathogenic bacteria while stimulating the growth of beneficial bacteria, exerting prebiotic and symbiotic (probiotic and prebiotic) effects. Interaction between functional food components, such as prebiotic, probiotic, phytochemicals and intestinal microbiota, have consequences on human health. There are various plant sources of nutraceuticals, including vegetables, fruits, seeds and spices available in

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nature. A balanced intake of vegetables, beans, fruit and grains can provide an array of beneficial compounds. Studies have demonstrated multiple effects of vegetables and fruits, for example, low concentration of fat, salt and sugar. Moreover, they are good source of dietary fiber. They contain vitamins and minerals good for health, including vitamins A (β-carotene), C and E, magnesium, folic acid, phosphorous and zinc [26]. Plants are rich sources of alkaloids, flavonoids and other beneficial components that can be specified as part of functional foods and nutraceuticals. It may be possible to alter the metabolic pathways and mechanisms to specifically enhance the production of desired metabolites. Various plant parts can be used on daily basis to serve as nutraceuticals and functional foods. Plants-based nutraceuticals and functional foods are safe to consume. Common examples of plant nutraceuticals include glucosamine from ginseng, Omega-3 fatty acids from linseed, Epigallocatechin gallate from green tea and lycopene from tomato [28]. Currently, scientists are working on nutraceuticals that can also be used for food preservation purposes.

10.3 Phytoremediation Phytoremediation may be defined as “application of plants for in situ, or in place, removal, degradation, or containment of contaminants in soil, sludge, sediments, surface water and groundwater.” It is very useful at sites with shallow and low levels of contamination. Phytoremediation is particularly useful for treating a wide variety of environmental contaminants. The following plant-mediated processes are useful for solving environmental problems: 1. Phytosequestration – It is an environment-friendly approach that uses plants to clean up soil from trace element contamination. It may be defined as the ability of plants to sequester certain contaminants in root zone. Various aquatic plants (Marsilea quadrifolia, Hydrilla verticillata and Ipomea aquatica) as well as terrestrial plants (Sesbania cannabina and Eclipta alba) have been found efficient accumulator of metals [29]. 2. Phytoextraction – It involves uptake and concentration of matter from the environment into the plant biomass. 3. Phytostabilization – It is the ability to reduce the mobility of specific substances in the environment, for example, by limiting the leaching of substances from the soil. 4. Phytotransformation – It is the chemical modification of environmental substances due to plant metabolism, which often results in their degradation (phytodegradation), inactivation or immobilization (phytostabilization). 5. Phytostimulation – It involves the enhancement of microbial activity of soil for degradation of contaminants by organisms that are associated with roots. This

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process is also known as rhizosphere degradation. Phytostimulation also involves aquatic plants supporting the active populations of microbial degraders, as in the stimulation of atrazine degradation by hornwort [30]. 6. Phytovolatilization – It is the process of removal of substances from soil or water and release into the air. Sometimes it is due to the phytotransformation to more volatile and/or less polluting substances. 7. Rhizofiltration – It is the process of water filtration through a mass of roots to remove toxic substances or excess nutrients. The pollutants remain absorbed in roots. Biological hydraulic containment through the plant decreases the movement of soluble contaminants deeper into the site and into the groundwater [31].

10.4 Plant molecular farming for industrial enzymes and therapeutic proteins Industrial enzymes are useful for food, feed, textile, paper, detergents, meat and energy industries. Various studies have demonstrated the feasibility of transgenic plants to accumulate industrial enzymes (alpha amylase, laccase, phytase) [32]. The heterologous gene expression is a good method to develop new pathways in plant and animals. The heterologous expression of α-amylase from Bacillus licheniformis was reported for the first time in tobacco plant [33]. This enzyme is commonly used in the food industry to produce high-fructose corn syrup, ethanol or detergents to remove starch [34]. The enzyme transglutaminase commonly used in the food industry has also been expressed in transgenic rice [35]. Glucosyltransferases (gtfD) expression from Streptococcus mutans in transgenic maize seed led to the accumulation of novel glucans, polymers with many applications in food industry, for example, as thickening agent [36]. In the last 30 years, transgenic plants have been predominantly utilized as a factory for the production of economically valuable recombinant proteins (also known as molecular farming). Several examples of molecular farming products are industrial enzymes, antibodies, antigens and vaccines [37, 38]. Plant system offers several advantages in terms of scalability, relatively low cost and free from human or animal pathogens, thus providing a safe environment [39]. Human growth hormone and interferon were among the first molecular farming products that have been successfully expressed in plants [40, 41]. Several plants-derived recombinant pharmaceuticals such as avidin [42–44] and beta glucuronidase [45] have been produced and commercialized. The various industrial enzymes and their applications produced in plant tissue culture and transgenic plants are amylases (food processing, paper industry), avidin and beta-glucuronidase (diagnostic kits), cellulases (ethanol and paper industry, dish washing detergents), glucanase (brewing industries), lignin peroxidase (paper industry), trypsin (pharmaceutical industry), pepsin (cheese industry), xylanses (biomass processing, paper and pulp industry) and phytase (better phosphate utilization).

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Plants have been used to manufacture drugs, antibodies and vaccines. One of the biopharmaceuticals, enkephalins, expressed in transgenic plant is useful as pain killer [46]. FDA has approved a drug taliglucerase alpha expressed by transgenic carrot cell culture for Gaucher disease treatment [47]. Recently, plant-derived products have been approved for commercialization or are in the clinical trials stage [39, 48, 49]. Plant derived antibody has been reported by researchers [50]. Accumulation and assembly of immunoglobulins in tobacco has been carried out successfully. More evidences regarding plant producing antibodies were reported afterward [39, 51, 52]. Arabidopsis-derived antibodies (IgA) designed for the passive immunization against enterotoxigenic Escherichia coli infection prolonged protection of weaned piglets from diarrhea [53]. In 2014, the plant antibodies were derived from tobacco to treat Ebola virus in human [54]. In order to prevent diseases, vaccines have to be quickly produced in enough quantity. Transient expression in plants offers production system of pharmaceuticals that can be achieved in 2–4 weeks [55]. Plant-derived vaccines also offer oral delivery that possibly removes the necessity of needle injection and the requirement of protein purification, and thus allows easy administration [56]. Oral administration also enhances and induces mucosal immunity, which is an important factor for stronger immune response. Plant-derived vaccines have been produced for pathogenic diseases such as hepatitis B, enterotoxin E.coli and influenza [57–59]. According to Sandhu et al. [60], orally fed mice with transgenic tomato expressing respiratory syncytial virus-F protein (RSV-F) stimulate the production of IgG and IgA antibodies indicating serum and mucosal immunity.

10.5 Conclusion The biotechnological techniques such as rDNA technology and metabolic engineering can be useful for altering or enhancing synthesis of various industrial important molecules and metabolites. The plant metabolites are safe to consume as compared to their chemical counterparts. Besides therapeutics, functional foods and nutraceuticals, the plants resources can also be used for environmental issues. Plant resources can also be used as production sites for pigments, colors, alkaloids, flavonoids, therapeutic agents, edible vaccines and phytoremediation. In coming years, more population will move toward the plants-based products and this will increase the demand of phytoproducts. Immense research and commercialization is required to satisfy the demand of plant products having no side effects, safe to consume and simultaneously avoiding social issues. Acknowledgement: The authors (RKP, PJ, SK, RK) are thankful to management of Ambala College of Engineering and Applied Research (ACE) Ambala, Haryana for encouraging us to write this informative manuscript.

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Index Abiotic tolerant 150 Acclimatization 127 Accumulation 90 Activity 95 Adulteration 116 Adventitious buds 135 Affinity chromatography 9 Agaricus bisporus 84 Agribusiness 136 Agricultural industry 147 Agro-industrial 62 Aloe vera 133, 211 Alzheimer’s disease 113 Amperometric 73, 76 α-amylase 15 Amylopectin 15 Amylopullulanases 15 Anaerobic digestion 58 Anti-arthritic 115 Antibiotic-resistant genes 182 Antibodies 74 Anticancer activity 98 Anticancerous 131 Anticarcinogenic 17 Anticoagulant factors 158 Antigenic sequences 159 Antigens 185 Anti-lipase 115 Antimicrobial 98 Anti-obesetic agents 113 Antioxidant 98, 209 Antioxidants 210 Apical meristems 128 Aromatic 137 Artemesinin 117 Ascorbic acid 83 Aseptic cultures 124 aseptic inoculation 80 Aspergillus niger 8 Auxins 126 Avocado 77 Axillary buds 123 Ayurveda 109, 133 Bacteria 14 Ball milling 55 Banana 136 https://doi.org/10.1515/9783110563337-011

Bananatrode 76 Bavistin 131 Bioactive compounds 96 Bio-based lubricants 161 Biobutanol 58 Biocatalyst(s) 9, 18, 37, 62, 77 Bio-component 75 Biodegradable plastics 186 Bioethanol 51, 57 Biofuel generation 128 Biofuel production 153 Biofuels 33 Biogas 58 Biohydrogen 57 Biolistic 179 Biomethane 58 Bio-optrode detection 81 Biopharmaceutical 154 Biopharmaceuticals 173, 185 Bioplastics 146 Bioreactors 137, 186 Biosafety 145 Biosensor 73 Biosynthesis 89 Biotransformation 130 Calendic acid 159 Callus 122 Cardiovascular diseases 209 Castor 159 Catharanthus roseus 130 Cell-disruption 37 Cellulases 39 Cellulose 51 Chemotherapy 113 Chimeric 186 Chitin matrix 79 Chloroplast engineering 176 Chloroplast transformation 174 Chloroplast transformation technology 176 Chloroplast transit peptide 192 Citric acid 64 Commercial aspects 205 Compressed gases 178 Consolidated biomass processing 56 Content 99 Coppiced shoots 129

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Crotonaldehyde dehydrogenation 58 CTT-derived cutinase 188 Cultivars 132 Cytokinins 126 Dark fermentation 61 Delignification 148 Demands 99 Dental caries 116 Diabetes mellitus 116, 210 Diacylglycerol acyltransferase 153 Diamine oxidase 82 Downstream processing 32 Edible vaccines 205 Elaioplasts 177 Embryo 131 Endangered 124 Endoglucanase 56 Endoinulinases 2 5-enolpyruvylshikimate-3-phosphate (EPSPS) 189 Entrapped 77 Enzymatic hydrolysis 33, 54 Enzyme 75 Enzyme kinetics 22 Enzyme-purification 37 Enzyme technology 31 Epinephrine 77 Eucalyptus 125 Exoinulinases 2 Explants 126 Extraction 79 Fabrication 74 Fermentative microorganisms 55 Filamentous fungi 2 Flavonoids 206 Food crops 148 Foreign proteins 180 Fourier-transform infrared spectroscopy 110 Fructooligosaccharides 14 Fumaric acid 64 Functional foods 211 Fungi 14 Gel-filtration chromatography 9 Genetic manipulation 148 Genetic modification 145, 159

Genetic variation 124 Genomics 146 Geobacillius 18 Germplasm 136 germplasm conservation 124 Gibberellic acid 134 Glucoamylases 1, 17 β-glucosidasegene 180 Glutathione 81 Glycine betaine 190 Glycogen 15 β-glycosidic linkage 52 GM crops 146 Good manufacturing practices 117 Green biotechnology 43 Guy’s 13 186 Haploids 136 Hemicellulose 52 Herbicide 78 Herbicide resistance 182 High fructose syrup 14 High-throughput 91 Horizontal gene transfer 182 Horticultural crops 136 Human immunoglobulin 157 Hydroquinone 78 Immobilized 73 Immunoglobulins 214 Indicators 76 Industrial enzymes 32, 213 Insulin 185 Inulinases 1 Inulinolytic enzymes 40 Inulin-rich plant 8 In vitro culture 80, 90, 129 In vitro propagation 125 Ion-exchange chromatography 9 Itaconic 62 Kluyveromyces 4 Laccase oxidase 84 Lactic acid 65 Lactobionic 62 Leucaena 150 Leukemia 85 Lignin 51

Index

Lignin biosynthetic 154 Lignins 148 Lignocellulosic 32, 153 Lignocellulosic waste 31 β-limit dextrin 15 Lipases 42 Low-cost agricultural waste 43 Luminescence 76 Luminescence signal 81 Luminescent 74 Lympho-sarcoma 85 Lysine decarboxylase enzyme 82 Maltotriose 15 β-mannanase 188 Mass-scale proliferation 125 Maternal inheritance 178 Medicinal herbs 105 Medicinal plant 89, 94 Medicinal plants 105 Metabolic engineering 90, 94 Metabolic flux 94 Metabolite 94 Metabolomics 91 Methanogenesis 61 Michaelis–Menten constant 9 Microbial fermentation 35 Micropropagation 205 Micro-propagation 122 Microshoots 133 Modern Medicine 109 Monoclonal antibodies 157 Mother plant 128 MSI- 99, 190 MS medium 129 Murashige and Skoog 134 Myrrh 109 Natural products 209 Neopullulanase-α-amylase 17 New World Syndrome 113 Nodal explants 131 Nontargeted integration 181 Nuclear-encoded polymerase 180 Nuclear genome 176 Nutraceutical 211 Octadecanoylsucrose 5 Oil-bearing plant 153

Oligosaccharides 56 Omega-3 fatty acids 212 Organic acids 62 Organomercurial 188 Ornithine decarboxylase 82 Osmoprotectants 190 Parthenium hysterophorous 62 Parvovirus 186 Pectinases 41 Pentoses 57 PGRs 126 Pharmaceutical 106 Pharmaceutical drugs 89 Pharmaceuticals 145 Pharmacognosy 106 Pharmacokinetic 117 Phosphinothricin 189 p-hydroxybenzoic acid (pHBA) 186 Physicochemical transducers 85 Phytochemicals 111 Phytoextraction 212 Phytomedicine 105, 206 Phyto-molecules 115 Phytoremediation 187, 206, 212 Phytosequestration 212 Phytostabilization 212 Phytostimulation 212 Phytotransformation 212 Phytovolatilization 213 Pichia stipitis 58 Piezoelectric 74 Plant-based bio-products 147 Plant-based materials 82 Plantbiomass-conversion 51 Plant biotechnology 205 Plantlets 130 Plant-manufactured pharmaceuticals 154 Plant metabolites 205 Plant pigment 206 Plants resources 214 Plastid-encoded polymerase 180 Podophyllotoxin 99 Podophyllum hexandrum 99 Pollen transmission 177 Polycistronic transcription units 178 Polyhydroxybutyric acid 180 Populus 128 Posttranscriptional 181

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Index

Prebiotic 211 Pretreatments 56 Primary metabolites 110 Proliferation 135 Propagation 136 Proplastids 177 Proteases 41 Proteinase inhibitor 150 Protoplast 132 psbA promoter 180 Pullulan 66 Pullulanases 2 rbcS genes 192 Recalcitrant 54 Redox mediator 79 Regeneration 132 Regulatory genes/enzymes 90 Rhizofiltration 213 Rooted plantlets 134 Roots 96 Saccharification 22, 55 Saccharomyces cerevisiae 57 Salt tolerance 176 Schizophyllan 65 Secondary metabolites 89, 106, 206 Seedling-raised plants 126 Shake-flask fermentations 5 Shikonin 97, 98 Shoot buds 130 Shoot elongation 130 Shoot multiplication 132 Shoot tip 134 Silymarin 117 Solid-state fermentation 4 Solubilization 78 Somatic embryogenesis 129 Starch 15 Stationary phase 110 Submerged fermentation(s) 1, 37 Succinic acid 64 Succinic acid-producing bacteria 65

Sulfite oxidase 84 Surface contaminants 133 Survival rate 127, 132 Sustainable agriculture 32, 44, 173 Swollenin 188 Symbiotic 211 Synergism 5 Teflon 83 Terpenoids 111 Therapeutic 137 Therapeutic agents 154 Thermal detection 74 Thermistors 75 Thidiazuron 129 Thin layer chromatography 109 Tissue culture 122 γ-tocopherol methyl transferase 191 Totipotency 122 Transcription factors 154 Transcriptomics 91 Transducer 73 Transgene cassette 181 Transgenic 146 Transgenic plants 213 Transgenic tobacco 178 Trp biosynthesis 187 Tryptophan 187 Understanding 91 Value-added products 32 Value addition 205 White-rot fungi 55 Withania somnifera 95, 96 Wood feedstock 125 Xylanas(es) 39, 187 Xylitol 40, 66 Xylose reductase 40 Yeast 14